Antisense oligonucleotides and uses thereof

ABSTRACT

Disclosed herein are novel compounds comprising antisense oligonucleotides that regulate the splicing of NUMB. In particular, an antisense oligonucleotide for reducing inclusion of NUMB exon 9 in a population of mature NUMB transcripts is provided. The antisense oligonucleotide comprises a sequence of at least 7 nucleotides that is complementary to a target region within exon 9 of a NUMB transcript. Pharmaceutical compositions comprising the antisense oligonucleotide and methods of treating a proliferative disease using the compounds or compositions of the invention are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/982,167, filed Sep. 18, 2020, which is a § 371 U.S. National StageApplication of International Application No. PCT/EP2019/056889 filedMar. 19, 2019, and claims priority to EP 18162571.6 filed Mar. 19, 2018,the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to antisense nucleotides that promote theexclusion of NUMB exon 9. In particular, the invention relates tooligonucleotides comprising a sequence that is complementary to a targetregion of a NUMB transcript, the target region being comprised in exon9. The oligonucleotides of the invention are particularly useful in thetreatment of proliferative diseases.

SEQUENCE LISTING

Applicant incorporates by reference a CRF sequence listing submittedherewith having file name FCD0001NA_Sequence.xml (248,131 bytes),created on Jun. 30, 2023. The nucleic acid and/or amino acid sequenceslisted in the accompanying sequence listing are shown using standardabbreviations as defined in 37 C.F.R. 1.822.

BACKGROUND OF THE INVENTION

Alternative splicing is a major regulatory mechanism in eukaryotic geneexpression, and its alterations have been linked with various diseases.For example, increased inclusion of a specific exon (exon 9) within theOpen Reading Frame of a protein called NUMB has been reported as one ofthe most common splicing alterations in lung cancer (Misquitta-Ali etal. 2011; Sebestyen et al., 2015). Increased NUMB exon 9 inclusion hasfurther been shown to increase the proliferative capacity of cancercells in vitro (Bechara et al., 2013). The NUMB protein is known to playa role in regulation of at least the Notch pathway: isoforms of NUMBthat exclude exon 9 act as repressors of the pathway and of cellproliferation, whereas isoforms of NUMB including exon 9 correlate withreduced NUMB protein levels and activation of the NOTCH pathway(Misquitta-Ali et al., 2011; Westhoff et al., 2009).

Because of the known correlation between NUMB alternative splicing ofexon 9 and cell proliferation, as well as data indicating that increasedNUMB exon 9 inclusion is amongst the most frequent tumour-associatedalternative splicing changes that have been observed in lung cancer, thealternative splicing of exon 9 of NUMB represents a potential target fortherapy. However, although some studies of alternative splicing havebeen conducted (see Bechara et al., 2013), the mechanisms involved arepoorly understood and there is still a need for new and more effectiveways of promoting the skipping of exon 9 in the NUMB pre-mRNA splicingprocess.

One potential strategy for regulating alternative splicing is the use ofantisense oligonucleotides (AONs). AONs are short synthetic nucleotideswhich can bind to a complementary target sequence. Therapies based onAONs have been developed and an AON that targets an intronic splicingenhancer site of the pre-mRNA corresponding to the protein SMN2(resulting in increased inclusion of exon 7), has been approved by theFDA and the EMA for the treatment of spinal muscular atrophy (Hua etal., 2010; Hua et al., 2011). However, efficient regulation of exon 9inclusion/exclusion in NUMB has not been achieved using AONs or othermethods.

The present invention seeks to provide compounds that can efficientlyregulate NUMB alternative splicing.

SUMMARY OF THE INVENTION

In general terms, the present invention provides new compoundscomprising oligonucleotides capable of regulating the alternativesplicing of exon 9 of NUMB.

In one aspect, therefore, the invention provides an antisenseoligonucleotide for reducing inclusion of NUMB exon 9 in a population ofmature NUMB transcripts, the antisense oligonucleotide comprising asequence of at least 7 nucleotides that is complementary to a targetregion within exon 9 (SEQ ID NO: 2 or homologues thereof) of a NUMBtranscript.

In a second aspect, the invention provides an antisense oligonucleotidefor reducing inclusion of NUMB exon 9 in a population of NUMB mRNAs, theantisense oligonucleotide comprising a sequence of at least 7nucleotides that is complementary to a target region within exon 9 (SEQID NO: 2 or homologues thereof) of a NUMB pre-mRNA.

According to a third aspect, an antisense oligonucleotide is disclosedcomprising a sequence of at least 7 nucleotides that is complementary toa target region of a NUMB transcript, wherein the target regioncomprises at least 7 nucleotides and is comprised in exon 9 (SEQ ID NO:2) or homologues thereof, and wherein, in use, the oligonucleotidereduces inclusion of exon 9 in the mature NUMB transcript.

According to a fourth aspect, there is provided an oligonucleotidecomprising 7 consecutive nucleotides of SEQ ID NO: 12, 22 or 24, whereinthe oligonucleotide binds to a target region of a NUMB transcript whichis comprised within NUMB exon 9, and wherein, in use, theoligonucleotide reduces inclusion of exon 9 in the mature NUMBtranscript.

In embodiments of any aspect, the antisense oligonucleotide comprises asequence selected from SEQ ID NO: 12, 13, 15-17 and 19-24, or homologuesthereof.

In embodiments, the target region is comprised within the sequence ofSEQ ID NO: 45-77, 306-316, 124-159 or 212-247 In embodiments, the targetregion is comprised within the sequence of SEQ ID NO: 78-104, 317-327,160-189, or 248-277. In embodiments, the target region is comprisedwithin the sequence of SEQ ID NO: 105-123, 328-338, 190-211 or 278-299.

In embodiments, the target region consists of the sequence of SEQ ID NO:45-77, 306-316, 124-159 or 212-247. In embodiments, the target regionconsists of the sequence of SEQ ID NO: 78-104, 317-327, 160-189, or248-277. In embodiments, the target region consists of the sequence ofSEQ ID NO: 105-123, 328-338, 190-211 or 278-299.

In embodiments, the target region is comprised within the sequenceCUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGC (SEQ ID NO: 9),CUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGCCAGCCCAUACU (SEQ ID NO:305),CCGUAGCAAUGCCUGUGCGUGAAACCAACCCUUGGGCCCAUG (SEQ ID NO: 10), orCCCCUGAUGCUGCUAACAAGGAAAUUGCAGCCACAUGUUCGG (SEQ ID NO: 11), orhomologues thereof. In some such embodiments, the oligonucleotide isfully complementary to a polynucleotide sequence comprised in SEQ ID NO:9, SEQ ID NO: 305, SEQ ID NO: 10, or SEQ ID NO: 11.

In embodiments, the target region is comprised within the sequenceCUAAUGGCACUGACUCAGCCU (SEQ. ID NO: 44), CCUGUGCGUGAAACCAACCCU (SEQ IDNO: 31), or GCUAACAAGGAAAUUGCAGCC (SEQ ID NO: 32), or homologuesthereof. In some such embodiments, the oligonucleotide is fullycomplementary to a sequence comprised in SEQ ID NO: 44, SEQ ID NO: 31,or SEQ ID NO: 32. In some embodiments, the oligonucleotide is fullycomplementary to a polynucleotide sequence comprised in SEQ ID NO: 44,SEQ ID NO: 31, or SEQ ID NO: 32.

In embodiments of any aspect of the invention, the antisenseoligonucleotide essentially consists of a nucleotide sequencecomplementary to a contiguous nucleotide sequence of a NUMB transcript.

In embodiments of any aspect of the invention, the antisenseoligonucleotide comprises a nucleotide sequence that is complementary toa target region comprising at least 13 nucleotides, at least 15nucleotides, at least 18 nucleotides or at least 21 nucleotides of NUMBexon 9.

In embodiments of any aspect of the invention, the antisenseoligonucleotide is between 7 and 31 nucleotides long. In embodiments,the antisense oligonucleotide is between 15 and 31 nucleotides long,between 18 and 31 nucleotides long, between 21 and 31 nucleotides long.In embodiments, the oligonucleotide is between 25 and 31 nucleotideslong.

In embodiments of any aspect of the invention, the antisenseoligonucleotide comprises a nucleotide sequence that is complementary toa target region of between 7 and 31 nucleotides, between 15 and 31nucleotides, between 18 and 31 nucleotides, between 21 and 31nucleotides, between 7 and 25 nucleotides, between 15 and 25nucleotides, between 18 and 25 nucleotides, or between 21 and 25nucleotides.

In embodiments, the antisense oligonucleotide is between 7 and 31nucleotides long, between 15 and 31 nucleotides long, between 18 and 31nucleotides long, between 21 and 31 nucleotides long, between 7 and 25nucleotides long, between 15 and 25 nucleotides long, between 18 and 25nucleotides long, or between 21 and 25 nucleotides long. In any of theembodiments of the invention, the oligonucleotide may be an RNA ormodified RNA molecule, a DNA or modified DNA molecule, or a mixture ofnative or modified RNA and native or modified RNA. One or more (e.g. 1,2, 3, 4, 5, 6 or more) nucleotides of the antisense oligonucleotide ofthe invention may be modified. For example, the oligonucleotide maycomprise a locked nucleic acid, a 2′-O-methyl phosphorothioateribonucleic acid, a 2′-O-methoxyethyl-modified phosphorothioateribonucleic acid, and/or one or more methylated cytosine residues.

In embodiments, the oligonucleotide is between 7 and 15 nucleotideslong, and the oligonucleotide is a locked nucleic acid.

In embodiments, the oligonucleotide is between 18 and 25 nucleotideslong, and the oligonucleotide is a 2′-O-methyl phosphorothioateribonucleic acid.

In embodiments, the oligonucleotide is between 18 and 25 nucleotideslong, and the oligonucleotide is a 2′-O-methoxyethyl-modifiedphosphorothioate ribonucleic acid. In embodiments, the oligonucleotideis between 18 and 25 nucleotides long, and the oligonucleotide is a2′-O-methyl-modified phosphorothioate ribonucleic acid.

In some embodiments, at least one cytosine residue within theoligonucleotide is methylated. In embodiments, all cytosine residueswithin the oligonucleotide are methylated.

In embodiments, the oligonucleotide does not comprise a sequence that iscomplementary to the 5′ and/or 3′ exon-intron junction of exon 9. Inembodiments, the oligonucleotide is fully complementary to apolynucleotide sequence within exon 9 of a NUMB transcript.

In embodiments, the oligonucleotide comprises a sequence selected from:AGGCUGA (SEQ ID NO: 36), GUCAGUG (SEQ ID NO: 37), CCAUUAG (SEQ ID NO:38), CUGAGUC (SEQ ID NO: 39), or AGUGCCA (SEQ ID NO: 40).

Suitably, the oligonucleotide may comprise a sequence selected from:AGGCUGAGUCAGUG (SEQ ID NO: 41), or GUCAGUGCCAUUAG (SEQ ID NO: 42).

In embodiments, the oligonucleotide comprises a sequence selected from:AGGCTGAGTCAGTGCCATTAG (SEQ ID NO: 43) or AGGCUGAGUCAGUGCCAUUAG (SEQ IDNO: 12).

In embodiments, the oligonucleotide comprises a sequence selected from:AGGCUGA (SEQ ID NO: 36), GUCAGUG (SEQ ID NO: 37), CCAUUAG (SEQ ID NO:38), CUGAGUC (SEQ ID NO: 39) or AGUGCCA (SEQ ID NO: 40). In someembodiments, the oligonucleotide consists of a sequence selected from:AGGCUGA (SEQ ID NO: 36), GUCAGUG (SEQ ID NO: 37), CCAUUAG (SEQ ID NO:38), CUGAGUC (SEQ ID NO: 39) or AGUGCCA (SEQ ID NO: 40).

In embodiments, the oligonucleotide comprises a sequence selected from:AGGCUGAGUCAGUG (SEQ ID NO: 41), or GUCAGUGCCAUUAG (SEQ ID NO: 42).Suitably, the oligonucleotide may consist of a sequence selected from:AGGCUGAGUCAGUG (SEQ ID NO: 41), or GUCAGUGCCAUUAG (SEQ ID NO: 42).

In embodiments, the oligonucleotide comprises a sequence selected from:AGGCTGAGTCAGTGCCATTAG or AGGCUGAGUCAGUGCCAUUAG (SEQ ID NO: 12).Suitably, the oligonucleotide may consist of a sequence selected from:AGGCTGAGTCAGTGCCATTAG or AGGCUGAGUCAGUGCCAUUAG (SEQ ID NO: 12).

The antisense oligonucleotide of any of the embodiments of the inventionmay be for use as a medicament.

Suitably, the antisense oligonucleotide may be for use in the treatmentof a disease or condition associated with elevated levels of NUMB exon 9inclusion in mature NUMB transcripts. In embodiments, the antisenseoligonucleotide may be for use in the treatment of a disease orcondition associated with elevated levels of NUMB exon 9 inclusion inmature NUMB transcripts compared to the level of NUMB exon 9 inclusionin a healthy matched tissue.

In embodiments, the antisense oligonucleotide of the invention may befor use in the treatment of a proliferative disease, such as a tumour ora cancer. In some such embodiments, the proliferative disease is adisease in which the numb protein acts as an antitumor agent.

In embodiments, the disease or condition is a proliferative disease inwhich the activation of the Notch pathway has a pro-proliferative role.In embodiments, the disease or condition is selected from lungadenocarcinoma, lung squamous-cell carcinoma, prostate cancer, cervicalcancer, breast cancer, pancreatic cancer, hepatocacinoma, osteosarcoma,neuroblastoma or colon cancer. In embodiments, the disease or conditionis lung adenocarcinoma, lung squamous-cell carcinoma, cervical cancer,breast cancer or colon cancer. In embodiments, the disease or conditionis selected from lung adenocarcinoma, lung squamous-cell carcinoma,cervical cancer, breast cancer, pancreatic cancer, prostate cancer,hepatocacinoma, or colon cancer.

In embodiments, the antisense oligonucleotide is for use in a method forthe treatment of a subject in need thereof, wherein the method comprisesadministering the oligonucleotide or a composition comprising theoligonucleotide to the subject.

Suitably, the method may further comprise testing a tissue sampleobtained from the subject for elevated levels of NUMB exon 9 inclusioncompared to a healthy control.

In embodiments, the subject is human.

In embodiments, the subject has a cancer or tumour and the methodcomprises delivering the oligonucleotide to the site of the cancer orthe tumour. In some such embodiments, the subject has lung cancer andthe method comprises delivering the oligonucleotide through therespiratory system.

In embodiments, the delivery through the respiratory system is performedintranasally, or via intratracheal administration. In embodiments, thedelivery through the respiratory system is performed by nebulisation orvia any other direct lung delivery system.

In embodiments, the method comprises injecting a composition comprisingthe oligonucleotide intravenously.

The invention is also directed, in another aspect, to a method oftreating a proliferative disease, the method comprising administeringthe antisense oligonucleotide of any of the preceding aspects to asubject in need thereof. In embodiments, the subject is human.

In embodiments, the subject has a cancer or tumour. Suitably, the methodmay further comprise delivering the oligonucleotide to the site of thecancer/tumour.

In embodiments, the subject has lung cancer and the method comprisesdelivering the oligonucleotide through the respiratory system. Inembodiments, the method comprises delivering the oligonucleotide throughthe respiratory system intranasally or intratracheally. In embodiments,the method comprises delivering the oligonucleotide by nebulisation.

In embodiments, the method comprises injecting a composition comprisingthe oligonucleotide intravenously.

In embodiments, the method further comprises testing the patient forelevated levels of NUMB exon 9 inclusion compared to a healthy control.

The invention, in another aspect, also encompasses a method of reducingNUMB exon 9 inclusion in a subject in need thereof, the methodcomprising administering the oligonucleotide of any of the first tofourth aspects to the subject.

In yet another aspect, the invention encompasses a method of reducingNUMB exon 9 inclusion in a population of NUMB mature mRNAs, the methodcomprising contacting a population of NUMB pre-mRNA with an antisenseoligonucleotide according to any of the first to fourth aspects.

According to a further aspect, the invention provides a pharmaceuticalcomposition comprising one or more antisense oligonucleotides accordingto any of the first to fourth aspects.

In embodiments, the pharmaceutical composition further comprises one ormore excipients.

It will be appreciated that any features of one aspect or embodiment ofthe invention may be combined with any combination of features in anyother aspect or embodiment of the invention, unless otherwise stated,and such combinations are envisaged and are intended to be directly andunambiguously disclosed herein, and to fall within the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated by way of the accompanying drawingsin which:

FIG. 1 shows the results of an investigation of the levels of NUMB exon9 inclusion in different tumors compared to matched healthy tissues;

FIG. 2 shows the design (top) and results (middle and bottom) of asystematic scanning of NUMB exon 9 using 21 nt long AONs; cellsexpressing a reporter exon 9 minigene were transfected with AONsaccording to the top panel, their RNA was collected after 24 hours andRT-PCR was performed with primers specific for detecting NUMB exon 9inclusion/skipping in the minigene; the middle and bottom panels showthe relevant region from polyacrylamide gels following electrophoreticseparation of the PCR amplified products, and the percent splice in(PSI) that correspond to the data;

FIGS. 3A and 3B show the effect on endogenous NUMB exon 9 inclusion inA549 lung adenocarcinoma cell line following transfection with threedifferent concentrations of two AONs according to the inventionalongside a control AON based on a randomised sequence of 21 nts;separate lung adenocarcinoma cell populations were transfected with oneof two AONs of the invention at three different concentrations; RNA wascollected after 24 hours and RT-PCR was performed with primers specificfor detecting NUMB exon 9 inclusion/skipping. FIG. 3A shows the relevantregion of the polyacrylamide gels following electrophoretic separationof the PCR amplified products, and FIG. 3B shows the percent splice in(PSI) that correspond to the data;

FIGS. 4A and 4B show the effect of an AON according to the invention onNUMB exon 9 skipping in lung adenocarcinoma mouse model cell lines. Fourdifferent lung adenocarcinoma KRAS-G12V-derived mice cell lines withdifferent p53 status were used (FIG. 4A) along with a human lungadenocarcinoma cell line (FIG. 4B);

FIG. 5 shows the effect of an AON according to the invention on colonyformation in lung adenocarcinoma mouse model cell lines (four differentlung adenocarcinoma KRAS-G12V-derived mice cell lines with different p53status were used) and a human lung adenocarcinoma cell line, at twodifferent concentrations: 100 nM (left), and 50 nM (middle) in the mousemodel cell lines, and a concentration of 100 nM in the human cell line(right);

FIG. 6 shows the results of a computational prediction of potentialbinding sites for known endogenous splicing regulators in the human NUMBexon 9 nucleotide sequence;

FIGS. 7A and 7B show the results of an analysis of the relationshipbetween QKI expression and exon 9 inclusion. FIG. 7A illustrates Westernblot results documenting increased expression of QKI; and FIG. 7B showsPSI (percent of spliced in-fraction of exon inclusion) values of NUMBexon 9 from a NUMB minigene in which the binding site of QKI in thetarget sequence of AON1 has been modified;

FIG. 8 shows the result of an analysis of the effect of intratrachealadministration of an AON of the invention to healthy mice; a significantreduction in NUMB exon 9 inclusion is observed in the lung 78 hoursafter administration;

FIGS. 9A and 9B show an experimental design (FIG. 9A) for testing theeffect of an AON of the invention on NUMB exon 9 inclusion in a geneticmouse model of KRAS-G12V driven non-small cell lung carcinoma, where theAON is administered intratracheally (FIG. 9A); and the results thereof(FIG. 9B);

FIGS. 10A, 10B and 10C show an experimental design for testing theeffect of an AON of the invention on tumour progression in a geneticmouse model of KRAS-G12V driven non-small cell lung carcinoma, where theAON is administered intranasaly (FIG. 10A); and the results thereof(FIGS. 10B and 10C);

FIGS. 11A, 11B and 11C show an experimental design for testing theeffect of an AON of the invention on NUMB exon 9 inclusion in a healthyadult mouse, where the AON is injected intravenously (FIG. 11A); and theresults thereof (FIGS. 11B and 11C, respectively showing the effect inthe liver and the lung);

FIGS. 12A and 12B show an experimental design for testing the effect ofintranasal administration of two different AONs of the invention in anorthotopic model of lung adenocarcinoma (FIG. 12A); and the resultsthereof (FIG. 12B);

FIG. 13 shows the effect of treatment with an AON according to theinvention on NUMB exon 9 inclusion in two different colon cancer celllines (HT29 and LS174T);

FIGS. 14A, 14B, 14C and 14D show the result of an analysis of the effectof an AON of the invention on colony formation in two different coloncancer cell lines (FIGS. 14A and 14B), a cervical cancer cell line (FIG.14C), and a hepatocarcinoma cell line (FIG. 14D);

FIGS. 15A and 15B show the effect on endogenous NUMB exon 9 inclusion(FIG. 15A) and colony formation (FIG. 15B) in A549 lung adenocarcinomacell line following transfection with an AON according to the inventionalongside a control AON based on a randomised sequence of 21 nts,wherein both the AON according to the invention and the control have amodified chemistry based on a locked nucleic acid (LNA) backbone;

FIG. 16 shows the effect on endogenous NUMB exon 9 inclusion in aHEK-293T cell line following transfection with 100 nM of AON1, accordingto the invention, alongside a control AON based on a randomised sequenceof 21 nts, wherein both the AON according to the invention and thecontrol have a modified chemistry based on a 2′-O-methyl modified AONwith a phosphorothioate backbone;

FIGS. 17A and 17B show the effect on endogenous NUMB exon 9 inclusion(FIG. 17A) and colony formation (FIG. 17B) in A549 lung adenocarcinomacell line following transfection with various AONs according to theinvention covering the region of NUMB exon 9 having the sequence of SEQID NO: 305, alongside a control AON based on a randomised sequence of 21nts;

FIGS. 18A and 18B show the effect on endogenous NUMB exon 9 inclusion inHEK-293T following transfection with various AONs of various lengthsaccording to the invention covering different parts of a specific region(Region 1) of NUMB exon 9, alongside a control AON based on a randomisedsequence of 21 nts, at a concentration of 100 nM (FIG. 18A) and 1,000 nM(FIG. 18B);

FIGS. 19A and 19B show the effect on endogenous NUMB exon 9 inclusion(FIG. 19A) and colony formation (FIG. 19B) in A549 lung adenocarcinomacell line following transfection with two AONs according to theinvention tested in FIGS. 18A and 18B, alongside a control AON based ona randomised sequence of 14 nts;

FIG. 20 shows schematically the sequences of the 3′ end of exon 9 ofhuman Numb, together with the nucleic acid binding/target sequenceswithin the NUMB gene of the AONs tested in FIGS. 17 to 19 (SEQ ID NO:358-372);

FIG. 21 shows the result of an analysis of the effect of an AON of theinvention (AON1) on colony formation in different cancer cell lines; and

FIG. 22 shows the result of an analysis of the effect of an AON of theinvention (AON6-7) on colony formation and exon 9 inclusion in differentcancer cell lines.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

Unless otherwise indicated, the practice of the present inventionemploys conventional techniques in chemistry and chemical methods,biochemistry, pharmaceutical formulation, and delivery and treatmentregimes for patients, which are within the capabilities of a person ofordinary skill in the art. Such techniques are also described in theliterature cited herein, each of which is herein incorporated byreference.

Prior to setting forth the detailed description of the invention, anumber of definitions are provided that will assist in the understandingof the invention.

The term ‘antisense oligonucleotide’ (AON) refers to a shortoligonucleotide or modified oligonucleotide which is antisense to andbinds to (i.e. hybridises to) a target region of a polynucleotide, suchas a gene transcript, pre-mRNA, mRNA or RNA fragment. According to theinvention, the target region for an AON is preferably in a pre-mRNA,i.e. the AON binds to a target region on a transcript before thesplicing process takes place. The AONs of the invention may comprisenative or modified RNA, DNA, or mixtures thereof. Any modification maybe naturally occurring or non-naturally occurring. Any references to anAON sequence provided as an RNA sequence is intended to also encompassthe equivalent DNA sequence. In particular, any sequence comprising Ubases is intended to refer equally to the corresponding sequence inwhich T bases are present in place of one or more (e.g. all) of the Us.In embodiments, the oligonucleotides according to the invention maycomprise nucleotides comprising inosine. In particular, any AON sequencedisclosed herein is intended to encompass nucleotide sequences where oneor more of the T, G or As are replaced with I, as far as this iscompatible with the function of the AON. The AONs according to theinvention have an effect on the regulation of alternative splicing. Inparticular, the AONs according to the invention advantageously promotethe skipping of a specific exon, resulting in a decreased inclusion ofthe exon in the mature transcript resulting from splicing of thepre-mRNA. As the skilled person would understand, the range of lengthsof AONs that are suitable will depend on the desired specificity (wherelonger AONs are expected to bind to their target sequence with higherspecificity) and efficacy of the in vivo delivery (where longer AONs areexpected to be more difficult to efficiently deliver to the targetcells). The AON's of the invention preferably have a length of at most35 nucleotides, at most 25 nucleotides, at most 24 nucleotides, at most21 nucleotides, or at most 18 nucleotides. An oligonucleotide as usedherein may have a length of at least 7 nucleotides, at least 10nucleotides, at least 13 nucleotides, at least 14 nucleotides, or atleast 17 nucleotides.

As known in the art, the AONs of the invention may be chemicallymodified, for example to increase their stability, reduce theirimmunogenicity, increase their binding affinity to a target sequence,reduce their non-specific binding to unintended targets (see e.g. Mou &Gray, 2002) and/or enhance their delivery, etc. Chemical modification ofan oligonucleotide refers to a chemical difference compared to thenative form of ribo or deoxyribonucelotides (i.e. nucleotides comprisingthe naturally occurring nucleobases of RNA or DNA, including(deoxy)adenosine, (deoxy)guanosine, (deoxy)thymidine, (deoxy)cytidine,5-methyl (deoxy)cytidine and uridine; and a phosphate linking group).Chemical modifications may be applied to the sugar moiety of anucleoside, the nucleobase moiety of a nucleoside and/or the phosphatebackbone. Examples of modified chemically modified nucleotides oranalogues that may be used in the present invention include lockednucleic acids, the use of a phosphorothioate backbone, 2′-O-methylatednucleotides, 2′-O-methoxyethyl modified (2′MOE) nucleotides, methylatedcytosine, constrained ethyl (cET) nucleic acids, bridged nucleic acids(BNAs), phosphorodiamidate morpholino oligomers (PMOs), peptide nucleicaids (PNAs), cyclohexene nucleic acids (CeNAs), tricycle-DNA (tcDNA),N3′-P5′ phosphoroamidates (NPs),Z-fluoro-Z-deoxyadenosine-5′-triphosphate,2′-fluoro-2′-deoxycytidine-5′-triphosphate,2′-fluoro-Z-deoxyguanosine-5′-triphosphate,2′-fluoro-2′-deoxyuridine-5′-triphosphate,2′-fluoro-Z-deoxythymidine-5′-triphosphate,2′-O-methyladenosine-5′-triphosphate,2′-O-methylcytidine-5′-triphosphate,2′-O-methylguanosine-5′-triphosphate, Z—O-methyluridine-g-triphosphate,2′-O-methylinosine-5′-triphosphate, 2′-O-methyl-2-aminoadenosine-5′-triphosphate, 2′-O-methylpseudouridine-5′-triphosphate,2′-O-methyl-5-methyluridine-5′-triphosphate etc. Chemical modificationsmay be applied to one or more nucleotides of an oligonucleotide and, assuch, reference to a ‘modified (antisense) oligonucleotide’ or ‘modifiedAON’ relates to oligonucleotides comprising at least one modifiednucleotide. In embodiments, 2′-0 modified nucleotides may be used tosurround (i.e. flank) a central sequence, thereby forming a gapmer, asknown in the art. Such compounds may be particularly resistant tonuclease degradation. As the skilled person would understand, multiplemodifications may be present on the same nucleotide. Further, differentmodifications may be present on different nucleotides of the sameoligonucleotide. As known in the art, the optimal length of an AON maydepend on the modifications (or absence thereof) that may be present onthe AON.

A ‘locked nucleic acid’ (LNA) as used herein refers to a ribonucleicacid where at least one of the nucleotides has the ribose moietymodified with a methylene bridge connecting the 2′ oxygen and the 4′carbon. Without wishing to be bound by theory, it is believed that thislocks the ribose ring in an ideal conformation for Watson-Crickbase-pairing, making the pairing of a locked nucleotide with acomplementary nucleotide strand more rapid and more stable. A lockednucleic acid may comprise a mixture of locked nucleotides andribonucleic acids. In embodiments, all of the nucleotides of theoligonucleotides according to the invention are locked nucleotides. Inembodiments where the AON is an LNA, the length of the AON may bebetween 7 and 21 nucleotides. In some embodiments, the length of the AONmay be between 7 and 17 nucleotides. In some such embodiments, thelength of the AON is 13 or 14 nucleotides.

As used herein a ‘phosphorothioate (ribo)nucleic acid’ (oroligonucleotide phosphorothioate) refers to a modified (ribo)nucleicacid in which one of the oxygen atoms in the phosphate moiety isreplaced by sulphur, wherein the oxygen that is replaced by sulphur isat a non-bridging position. Without wishing to be bound by theory, it isbelieved that because phosphorothioate (ribo)nucleic acids arenon-natural analogs of nucleic acids, oligonucleotide phosphorothioatesare substantially more stable with respect to hydrolysis by nucleases,the class of enzymes that destroy nucleic acids by breaking the bridgingP—O bond of the phosphodiester moiety. An oligonucleotidephosphorothioate may comprise a mixture of modified and unmodifiednucleotides. In embodiments, the whole backbone of the oligonucleotideof the invention is modified, and each nucleotide may be aphosphorothioate nucleotide.

AONs according to the invention may comprise 2′-O-methylatedribonucleotides. 2′-O-methylated ribonucleotides comprise a methyl groupadded to the 2′ hydroxyl of the ribose moiety of the nucleotide,producing a methoxy group. In embodiments, AONs according to theinvention may comprise one or more 2′-O-methylated ribonucleotides. Inembodiments, all of the nucleotides of the AON may be 2′-O-methylatedribonucleotides.

AONs according to the invention may comprise 2′-O-methoxyethylribonucleotides. 2′-O-methoxyethyl ribonucleotides comprise amethoxyethyl group (—CH₂CH₂OCH₃) added to the 2′ hydroxyl of the ribosemoiety of the nucleotide. In embodiments, AONs according to theinvention may comprise one or more 2′-O-methoxyethyl ribonucleotides. Inembodiments, all of the nucleotides of the AON may be 2′-O-methoxyethylribonucleotides.

In some embodiments the AONs according to the invention may comprise amixture of one or more ribonucleotides modified with a methyl group(e.g. 2′-O-methylated ribonucleotides) and one or more ribonucleotidesmodified with a methoxyethyl group (e.g. 2′-O-methoxylethylribonucleotides).

In embodiments, the oligonucleotides may have the same or similarchemical modifications as the AON therapy Spinraza (Nusinersen, RNA,(2′-O-(2-METHOETHY I))(P-THIO)(M5U-C-A-C-M5U-M5U-M 5U-C-A-M5UA-A-M5U-G-C-M5U-G-G), CAS1258984-36-9).

In embodiments, the oligonucleotides according to the invention maycomprise one or more conjugate groups, for example, in order to modifythe properties of the compound, such as the pharmacodynamics,distribution, stability, binding and/or absorption etc. of the compound,as known in the art. In embodiments, the one or more conjugate groupsmay comprise a ligand that targets the compound, for example to aspecific type of cells. Thus, in embodiments, the invention encompassescompounds comprising one or more oligonucleotides according to theinvention fused to another chemical entity, such as a pharmaceuticaldrug. The conjugated entity (or group) may comprise nucleotides or maybe a non-nucleotide based molecule. In embodiments, the conjugatedentity may comprise a splicing modifying drug. Examples of splicingmodifying drugs are provided in Vigevani, L., & Valcarcel, J. (2012). Inembodiments, the drug is selected from the group comprising sudemycins,spliceostatin, pladienolides and meayamycins, or derivatives thereof.

References to a reduction in exon 9 inclusion or promotion of exon 9skipping throughout this disclosure are equivalent and refer to areduced percentage of NUMB transcript spliced to include exon 9(expressed as ‘percent spliced in’/‘percentage splicing index’ PSI)compared to a control condition. Conversely, references to an increasein exon 9 inclusion, or inhibition/reduction of exon 9 skipping, referto an increased percent spliced in (PSI) of exon 9 compared to a controlcondition. PSI may be obtained using methods known in the art, includingreverse transcription-polymerase chain reaction (RT-PCR), mRNAsequencing (RNA-seq), etc.

The inventors have surprisingly discovered that NUMB exon 9 inclusioncan be efficiently down-regulated by targeting specific regions withinexon 9 of NUMB with AONs. Specific regions within exon 9 of NUMB wereidentified as targets for AONs, and the AONs of the inventionbeneficially result in reduced inclusion of exon 9 in NUMB transcripts.Further, the inventors have surprisingly discovered that targetingspecific regions within exon 9 of NUMB with AONs can result in moreefficient down-regulation of exon 9 inclusion than is possible bytargeting the splice sites of exon 9.

As used herein, references to exon 9 of NUMB relate to positions 1417 to1560 (SEQ ID NO: 2) of Human Numb mRNA transcript variant 1(NM_001005743) or equivalent positions in other transcript variantsencoded by the NUMB gene (Entrez Gene ID 8650), or homologues thereof(such as the mouse homolog of SEQ ID NO: 1 (positions 1115 to 1261 ofNM_001136075.2 —Mus Musculus Numb mRNA transcript variant 1).

The inventors have discovered that targeting specific regions withinexon 9 of the NUMB transcript with AONs resulted in a significantdecrease in the inclusion of exon 9 in mature transcripts of NUMB. Inparticular, AONs according to the invention may comprise a sequence thatis complementary to a target sequence in specific regions within NUMBexon 9, the target sequence being at least 7 nucleotides long and beinglocated within the regions defined by SEQ ID NO: 9, 305, 10 or 11; orhomologues thereof. In other words, the AONs according to the inventionmay comprise a sequence that is complementary to at least 7 contiguousnucleotides located within one of the regions underlined in thesequences below, or homologues thereof.

Human NUMB exon 9 (positions 1417 . . . 1560 of NM_001005743; SEQ ID NO:2): cta atggcac tgactca gcc ttccatgtgc ttgctaagcc agcccatact gctctagcacccgtagcaat gcctgtgcgt gaaaccaacc cttgggccca tg cccctgat gctgctaacaaggaaattgca gccacatgtt cgg

Mouse NUMB exon 9 (positions 1115 to 1261 of NM_001136075.2; SEQ ID NO:1): cta a tggcac tgactca gcc tcccatgtgc ttactgctaa gccagccaatactgctctagc acacgtagca atgcctgtcc gtgaaa ccaa cccctgggcc catgtccctgatgctgctaa caaggaaatt gcagccatac atccgg

In embodiments, the target sequence may be located within subregions ofthe regions underlined above. In particular, in embodiments the targetregion may be located within the region defined by sequence SEQ. ID NO:44. In other embodiments, the target regions may be located within theregion defined by sequence SEQ. ID NO: 31. In embodiments the targetregion may be located within the region defined by sequence SEQ. ID NO:32. In other words, the AONs according to embodiments of the inventionmay comprise a sequence that is complementary to at least 7 nucleotideslocated within one of the regions in bold in the sequences above, orhomologues thereof.

In embodiments, the target sequence (also referred to herein as “targetregion”) may be 7 nucleotides long and may be defined by SEQ ID NO:45-77, 306-316, 124-159 or 212-247. In embodiments, the target sequencemay be 13 nucleotides long and may be defined by SEQ ID NO: 78-104,317-327, 160-189, or 248-277. In embodiments, the target sequence may be21 nucleotides long and may be defined by SEQ ID NO: 105-123, 328-338,190-211 or 278-299.

In embodiments, the target sequence (also referred to herein as “targetregion”) may be located within any of the regions defined by sequenceSEQ ID NO: 45-77, 306-316, 124-159 or 212-247. In embodiments, thetarget sequence may be located within any of the regions defined bysequence SEQ ID NO: 78-104, 317-327, 160-189, or 248-277. Inembodiments, the target sequence may be located within any of theregions defined by sequence SEQ ID NO: 105-123, 328-338, 190-211 or278-299.

In embodiments, AONs according to the invention may comprise 7nucleotides that are complementary to any of the sequences of SEQ ID NO:45-77, 306-316, 124-159 or 212-247. In embodiments, AONs according tothe invention may comprise 13 nucleotides that are complementary to anyof the sequences of SEQ ID NO: 78-104, 317-327, 160-189, or 248-277. Inembodiments, AONs according to the invention may comprise 21 nucleotidesthat are complementary to any of the sequences of SEQ ID NO: 105-123,328-338, 190-211 or 278-299.

In embodiments, the target region may comprise 7 nucleotides; or morethan 7 nucleotides, such as 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or 21 nucleotides that are located within any of the regions underlinedand optionally those regions shown in bold above. For example, thetarget region may comprise 13 nucleotides located within the regionsdefined by any of sequences SEQ ID NO: 9, 10, 11, 305, 44, 31 or 32. Inother embodiments, the target region may comprise 18 to 21 nucleotideslocated within the regions defined by any of sequences SEQ ID NO: 9, 10,11, 305, 44, 31 or 32. In other words, AONs according to the inventionmay comprise 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides that are complementary to 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or 21 nucleotides located within any of the regionsunderlined/in bold above. For example, AONs according to the inventionmay comprise 13 nucleotides that are complementary to 13 nucleotideslocated within the regions defined by any of sequences SEQ ID NO: 9, 10,11, 305, 44, 31 or 32. In other embodiments AONs according to theinvention may comprise 18 to 21 nucleotides that are complementary to 18to 21 nucleotides located within any of sequences SEQ ID NO: 9, 10, 11,305, 44, 31 or 32.

In embodiments, the AONs of the invention are suitably complementary toa portion of the sequence of a NUMB transcript. In such embodiment, theAONs may comprise a portion of sequence that is complementary to atarget region of at least 7 nucleotides as explained above, and aportion of sequence that is complementary to a region of the NUMBtranscript that does not lie within the regions described above. Forexample, an AON according to the invention may comprise a sequence thatis complementary to the last 7 nucleotides of sequence SEQ ID NO:9 (orSEQ ID NO: 10, 11, 44, 31, 32), and additional nucleotides complementarywith the part of the sequence of exon 9 that follows SEQ ID NO: 9 (i.e.SEQ ID NO: 4). Alternatively, the AONs may have a sequence such thatevery nucleotide of the AON is complementary with a nucleotide that lieswithin one of the regions identified above. For example, the AONs mayhave a sequence such that every nucleotide of the AON is complementarywith a nucleotide of SEQ ID NO: 9 (or SEQ ID NO: 10, 11, 44, 305, 31, 32or 33). Suitably, the sequences of the AONs of the invention arecomplementary to a sequence that is comprised within NUMB exon 9 (SEQ IDNO: 1, 2) or homologues thereof.

In embodiments, the target sequence may be located within subregions ofthe regions underlined above. In particular, in embodiments the targetregion may be located within the region defined by sequence SEQ. ID NO:44, 3, 5-7, 9, 31-35. In embodiments, the antisense oligonucleotidesaccording to the invention comprises a sequence selected from SEQ ID NO:12, 13, 15-17 and 19-24, or homologues thereof.

Without wishing to be bound by theory, it is believed that the AONs ofthe invention will be particularly useful in the treatment of diseasesor conditions that are associated with elevated levels of NUMB exon 9inclusion compared to a healthy matched tissue. In particular, it isbelieved that the AONs of the invention will be particularly useful inthe treatment of proliferative diseases where the NUMB protein acts toreduce cellular proliferation, since the isoform of NUMB that excludesexon 9 is associated with higher expression levels and reduced cellularproliferation, potentially via reduced activation of the Notch pathwayor other cellular proliferation pathways. For example, the AONs of theinvention have been shown (see Examples below) to have a beneficialeffect in preventing cellular proliferation in multiple cancer celllines, and to have beneficial effects on tumour progression (such as forexample reversing tumour progression) in vivo and/or in vitro inmultiple cancers. In particular, the AONs of the invention may be usefulin the treatment of lung cancers (in particular lung adenocarcinomas),cervical cancer, breast cancer, colon cancer, brain glioblastoma,pancreatic cancer, acute monocytic leukemia, kidney cancer, colorectalcancer, liver cancer (e.g. hepatocarcinoma), and glioblastoma. The AONsof the invention may also be useful in the treatment of skin cancer(e.g. melanoma), stomach cancer, thyroid cancer and bone cancer. Assuch, the invention provides compounds and compositions for use inmedicine and, in particular, for use in the treatment of cancersselected from lung cancers (in particular lung adenocarcinomas or lungsquamous carcinoma), bladder cancer, cervical cancer, breast cancer,colon cancer, brain glioblastoma, pancreatic cancer, acute monocyticleukemia, kidney cancer, colorectal cancer, skin cancer (e.g. melanoma),stomach cancer, thyroid cancer and bone cancer. Methods for thetreatment of such diseases are also provided. The uses and methods maycomprise administering the AONs according to the invention to a patientin need thereof.

In embodiments, the invention provides pharmaceutical compositionscomprising one or more AONs according to the invention. In embodiments,the compositions may comprise one or more pharmaceutically acceptableexcipients. For example, the compositions according to the invention maycomprise one or more AONs according to the invention and a buffer, suchas water or a saline buffer (e.g. phosphate-buffered saline, PBS,preferably pharmaceutical grade PBS). In embodiments, the compositionsmay comprise solid or gel excipients, such as flavouring agents,thickeners, stabilisers, carriers, diluents, surfactants, penetrationenhancers, emulsifiers and the like. For example, compositions accordingto the invention may comprise polyethylene glycol, gelatin, lactose,talc, silic acid, hydroxymethylcellulose, sodium carboxymethylcellulose,sorbitol, dextran, etc. In embodiments, a pharmaceutical compositionaccording to the invention may comprise a delivery system, such as aliposome.

In embodiments, a pharmaceutical composition according to the inventionmay comprise one or more excipients selected from the group comprising:NaCl and/or HCl aqueous solutions; tartaric acid in aqueous solution,methyl parahydroxybenzoate in aqueous solution; polysorbate 20(polyoxyethylene (20) monolaurate sorbitan monolaurate); dihydratedmonosodic phosphate; anhydrous disodic phosphate in aqueous solution;sodium methylparahydroxybenzoate; propylparahydroxybenzoate in aqueoussolution; EDTA/NaOH at pH 6.5 in aqueous solution; EDTA/NaOH at pH 7.5in aqueous solution; EDTA, NaCl, polysorbate 80, citric acid, Na citratein aqueous solution; NaCl and CaCl in aqueous solution; NaCl, H2504 andNaOH at pH 7 in aqueous solution; trometamol, ethanol 96%, NaCl, HCl inaqueous solution; trometamol, ethanol 96%, NaCl, HCl in aqueoussolution; sorbitan trioleate; menthol; norflurane (HFA-134a), Oleicacid, citric acid, HFA-134a, glycerol; cetylpyridinium chloride,sorbitan trioleate; norflurane; HFA-227; polyvinylpyrrolidone K30,polyethylene glycol 600; norflurane sorbitan; trioleate, magnesiumstearate; sugars (e.g. lactose, glucose, mannitol, trehalose, etc.); Mgstearate, lipids (such as DPPC, DSPC, DMPC, cholesterol, etc.); aminoacids (e.g. leucine, trileucine); surfactants (e.g. poloxamer); bilesalts; absorption enhancers (e.g. hydroypropylated-β-CD, natural γ-CD);chitosan; trimethylchitosan; and biodegradable polymers (e.g. PLGA).

In embodiments, a pharmaceutical composition according to the inventionmay comprise one or more solvents or cosolvants, such as organicsolvents (acetone, acetic acid, acetonitrile, benzene, carbontetrachloride, methylene chloride etc.) and inorganic solvents (liquidammonia, liquid sulfur dioxide, sulfuryl chloride and sulfuryl chloridefluoride, phosphoryl chloride, dinitrogen tetroxide, antimonytrichloride, bromine pentafluoride, hydrogen fluoride, pure sulfuricacid etc.) In embodiments, a pharmaceutical composition according to theinvention may comprise one or more propellants, such astrichloromonofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethane, or other chlorofluorocarbon propellants;chlorodifluoromethane, trifloromonofluoroethane, chlorodifluoroethane,difluoroethane, heptafluoropropane, or other hydrochlorofluorocarbon andhydrofluorocarbon propellants; propane, isobutene, butane, pentane orother hydrocarbon propellants; nitrogen, nitrous oxide, carbon dioxide,compressed air or other compressed gases propellants. In embodiments, apharmaceutical composition according to the invention may comprise oneor more preservatives, such as benzalkonium chloride, cetrimoniumbromide, benzethoniun chloride, alkyltrimethylannonium bromide,EDTA/Benzalkonium chloride, benzoic acid, soric acid, potassium sorbate,propylene glycol, ethanol, sodium benzoate, thimerosal, benzyl alcohol,chlorhexidine, chloroactamide, trichlorocarban, 4-chlorocresol,4-chloroxylenol, dichlorophene, hexachlorophene; methyl, ethyl, propyl,butyl Parabens and combinations thereof. In embodiments, apharmaceutical composition according to the invention may comprise oneor more humectants, such as propylene glycol, glycerol, polyethyleneglycol. In embodiments, a pharmaceutical composition according to theinvention may comprise one or more anti-foaming agents, such asinsoluble oils, polymethylsiloxanes and other silicones, stearates,glycols or polydimethylsiloxane-silicon dioxide. In embodiments, apharmaceutical composition according to the invention may comprise oneor more wetting agents, such polysorbates (e.g. Tweens), sorbitan esters(Spans), polysorbates, polysorbates, poloxamers, lecithin, soyalecithin, sodium lauryl sulphate, or hydrophilic colloids such asbentonite, tragacant, alginates, and cellulose derivatives. Inembodiments, a pharmaceutical composition may comprise ananoparticle-based drug formulation/delivery mechanism.

As the skilled person would understand, the appropriate mode ofadministration of the compounds and compositions of the invention mayvary depending on whether local or systemic administration is desired,as well as depending on the area to be treated. In embodiments,administration may be topical, pulmonary, oral or parenteral. Inembodiments, the compounds and compositions of the invention may beadministered orally, ophthalmically, intranasally (e.g. via an inhaler,insufflation of powders or aerosols, using a nebuliser),intratracheally, by injection (e.g. intravenous, intraarterial,subcutaneous, intramuscular, intraperitoneal, intramuscular,intracranial, etc.), topically or transmucosally (e.g. using transdermalpatches, ointments, lotions, suppositories, sprays, liquids, powders orgels). In embodiments, a pharmaceutical composition according to theinvention comprises one or more AONs in a therapeutically effectiveamount. Similarly, methods of treatment of a disease or conditionaccording to the invention may comprise administering the compounds orcompositions of the invention in a therapeutically effective amount. Inembodiments, a therapeutically effective amount is sufficient to preventor alleviate the symptoms of a disease, reduce the size or the rate ofprogression of a tumour or cancer, and/or prolong the survival of thesubject being treated. Determination of a therapeutically effectiveamount can be performed as known in the art, and may depend on the AONused, the method and frequency of administration, the disease treated,and/or the subject treated, etc.

In embodiments, methods of treatment of a subject using the compoundsand compositions of the invention may comprise repeated administrationof the compounds and compositions of the invention, as known in the art.In embodiments, methods of treatment of a subject using the compoundsand composition of the invention may comprise administration of anantiproliferative drug in combination with the compounds andcompositions of the invention, either sequentially, simultaneously orseparately. In embodiments, methods of treatment of a subject using thecompounds and compositions of the invention may comprise administrationof a splicing modifying drug in combination with the compounds andcompositions of the invention. ‘In combination with’, in the context ofthe invention, may mean that the two (or more) compounds may beadministered either sequentially, simultaneously or separately, asdesired.

In embodiments, methods of treatment of a subject using the compoundsand composition of the invention may comprise testing the subject forelevated levels of NUMB exon 9 inclusion compared to a healthy subjectand/or healthy tissue. For example, the methods of treatment accordingto the invention may comprise the steps of obtaining a sample (e.g. bybiopsy) of a diseases tissue (e.g. a tumour), and quantifying theinclusion of NUMB exon 9 compared to a matched tissue from a healthysample. Healthy and/or diseased samples may be liquid biopsy samples. Inembodiment, the healthy sample may have been obtained from healthytissue of the subject. In other embodiments, the healthy sample may havebeen obtained from healthy tissue of another subject. In yet otherembodiments, the healthy sample may be a reference sample (e.g. a cellline). Quantifying the inclusion of NUMB exon 9 may be performed asknown in the art, for example by performing RT-PCR, RNA sequencing, etc.and calculating the PSI for NUMB exon 9. The methods may furthercomprise administering the compounds or compositions of the invention ifthe data obtained at the previous step indicate that the diseased tissueshows elevated levels of NUMB exon 9 inclusion.

The invention will now be further illustrated by way of the followingnon-limiting examples.

EXAMPLES

Unless otherwise indicated, commercially available reagents and standardtechniques in biology, chemistry and biochemistry were used.

Example 1: Exon 9 Inclusion is Increased in Many Different Types ofTumors

FIG. 1 shows the results of an investigation of the level of NUMB exon 9inclusion (PSI=percent spliced in, i.e. proportion of transcripts whereexon 9 is present compared to total number of transcripts for a givengene) in paired tumour/healthy samples for 11 tumour types using datafrom the TCGA consortium. The data shows that in at least BreastInvasive Carcinoma, Liver Hepatocellular Carcinoma, Lung Adenocarcinoma,Lung Squamous Cell Carcinoma and Prostate Adenocarcinoma, thedistribution of PSI for exon 9 of NUMB across samples is significantlyhigher in the tumour samples than in the healthy samples (Mann Whitney Utest, corrected for multiple testing using the Benjamini-Hochbergmethod). This indicates that regulation of NUMB exon 9 inclusion may bea potential avenue for therapy for many cancer types, in addition tolung adenocarcinoma.

Example 2: Identification of AONs Inducing NUMB Exon 9 Skipping

Having previously established that NUMB exons 9 inclusion could bemodulated on lung cancer A549 cells using 2′-O-methylphosphothioate-modified antisense oligonucleotides (AONs) complementaryto a region of the pre-mRNA encompassing the 5′ splice site (SS)(Bechara et al., 2013), the present inventors set out to identify newtarget regions that could be targeted by AONs to promote exon 9 skippingmore efficiently.

A systematic scanning of NUMB exon 9 was performed using 21 nucleotideAONs complementary to consecutive, overlapping regions of the exon: atotal of 24 AONs covering the 144 nucleotide exon were used in thisstudy. AONs complementary to regions of the pre-mRNA encompassing the 3′SS or the 5′ SS, the branch point (BP) region or a random sequence (RND)were included as controls.

FIG. 2 shows the results of this analysis. As can be seen in FIG. 2 ,cells transfected with the random AON show almost full exon 9 inclusion(PSI=0.93). Targeting the branch point region with the AON of SEQ ID NO:29 also had a very limited effect. By contrast, transfection with any ofthe AONs complementary to exon 9 (AON1 to AON7, which correspond to SEQID NO:12 to 18, respectively, and AON1-2, 2-3, 3-4, 4-5, 5-6 and 6-7corresponding to SEQ ID NO:19 to 24) resulted in significant exon 9skipping. In some cases, surprisingly, the level of exon skipping thatcan be obtained by targeting sequences within exon 9—in accordance withthe invention—is higher than that which can be obtained using AONs thattarget the splice sites of exon 9 (i.e. AONs of SEQ ID NO: 25 and 26,respectively for the 5′ and 3′ splice sites—targeting the sequences ofSEQ ID NOs: 27 and 28, respectively).

In particular, three regions of exon 9 were identified which, whentargeted by AONs of the invention, resulted in particularly efficientexon 9 skipping: these three regions are the combined regions targetedby AON1, AON2, AON1-2 and AON2-3 (i.e. the region of exon 9 having thesequence of SEQ ID NO: 305), and in particular the combined regionstargeted by AON1 and AON2 (also including the region targeted by AON1-2;i.e. the region of exon 9 having the sequence of SEQ ID NO: 9); thecombined regions targeted by AON4, AONS and AON4-5 (i.e. the region ofexon 9 having the sequence of SEQ ID NO:10); and the combined regionstargeted by AON6 and AON6-7 (i.e. the region of exon 9 having thesequence of SEQ ID NO: 11). When each of these regions was targeted byAONs, the PSI for exon 9 was reduced to 0-0.02 for the region of exon 9having the sequence of SEQ ID NO: 305 (targeted by AON1, AON2, AON1-2and AON2-3; i.e. SEQ ID NO: 12, 13, 19 and 20); 0-0.17 (region of exon 9having the sequence of SEQ ID NO:10 (targeted by AON4, AON5 AON4-5; i.e.SEQ ID NO:14, 15 and 22); and 0.05-0.55 (region of exon 9 having thesequence of SEQ ID NO: 11 (targeted by AON6, AON7, AON6-7; i.e. SEQ IDNO: 16, 17 and 24). Within those regions, subregions targeted by AON1(SEQ ID NO: 12), AON1-2 (SEQ ID NO: 19), AON4-5 (SEQ ID NO: 22) andAON6-7 (SEQ ID NO: 24) in particular—respectively the regions of exon 9having the sequences of SEQ ID NO: 30, 31 and 32—resulted in almostcomplete exon 9 skipping when targeted by AONs.

This data indicates that the targeting of specific regions within exon 9of NUMB with AONs results in effective skipping of exon 9 in full lengthNUMB transcripts.

Example 3: AONs According to the Invention are Efficacious at LowConcentration and on Endogeneous NUMB Transcripts

Investigations were carried out to determine whether the beneficialregions identified in the studies of Example 2 could be used to regulateexon 9 inclusion in endogeneous NUMB transcripts in a lungadenocarcinoma cell line (A549), and whether any such regulation wouldbe concentration-dependent. The A549 lung adenocarcinoma cell linedisplays high levels of NUMB exon 9 inclusion and NUMB splicing is knownto control cell proliferation in this cell line (Bechara et al., 2013).

Three different concentrations (5, 10 and 50 nM) of AON 1 (SEQ ID NO:12) and AON 4-5 (SEQ ID NO: 22) were transfected into A549 cells and, 24hours after transfection, RNA was isolated and the endogenous levels ofNUMB exon 9 inclusion/skipping were measured by RT-PCR. A random AON(RND) was used as a control.

FIGS. 3A and 3B show the results of this analysis. The data of FIG. 3A(polyacrylamide gels following electrophoretic separation of the RT-PCRproducts above, quantified on FIG. 3B) shows that titrable effects onexon skipping can be detected for both AON1 and AON 4-5 even at 5 nM,which progressively increases at higher AON concentrations. By contrast,no effect was detected using the random AON negative control. Note thatat 50 nm, NUMB transcripts without exon 9 represent the majority of theNUMB transcripts in cell culture (i.e. PSI <0.5). These differences inPSI are significant (Students t-test, *P-value <0.05, **P-value <0.005,***P-value <0.001) despite the fact that the transfections are extremelyunlikely to result in 100% of the cells being transfected. As such, someof the cells in the culture would be non-transfected and so the PSI incells that were transfected is expected to be lower than the global PSIthat was measured here.

Example 4: AONs According to the Invention Reduce Exon 9 Inclusion inLung Adenocarcinoma Mouse Models and a Human Lung Adenocarcinoma CellLine

The effect of AON1 (SEQ ID NO: 12), which targets a sequence of NUMBexon 9, which is consented between human and mouse, was tested in fourdifferent lung adenocarcinoma KRAS-G12V-derived mice cell lines (KLC1 to4, Ambrogio et al., 2014).

AON1 or a random control was transfected at a concentration of 50 nM ineach of the four cell lines. RNA was isolated 24 hours aftertransfection, NUMB mRNA was PCR amplified and the levels of NUMB exon 9inclusion/skipping were measured by capillary electrophoresis. Theresults of this analysis are shown on FIG. 4A. This figure demonstratesthat although NUMB exon 9 skipping is very prominent in these cells, theAONs further significantly decrease PSI values in almost all cell lines(apart from KLC4). However, the effect is more prominent in p53−/− cellscompared to p53+/+ cells (compare KLC1, KLC2 KLC3, KLC4). These datashow that the effect is reproducible in mouse lung adenocarcinomamodels, even when the level of exon 9 inclusion is lower, and also thatthe effect is not p53 dependent, although a stronger effect is observedin cells deficient for p53.

Similar results were obtained with a human lung adenocarcinoma cell line(A549), as shown on FIG. 4B.

Example 5: AONs According to the Invention Reduce Colony Formation inLung Adenocarcinoma Mouse Models and Human Cell Lines

The colony formation potential after transfection with AON1 was testedon the same cell lines as in Example 4, as well as the human lungadenocarcinoma cell line A549. Two concentrations of AON1 were tested:100 nM (left panel and right panel on FIG. 5 ), and 50 nM (middle panelon FIG. 5 ) in biological triplicates. The results of this analysis areshown in FIG. 5 . Note that the KCL4 cell line has limited clonogeniccapacity even under control conditions. The results indicate that thereduction in NUMB exon 9 inclusion correlates with a reduced potentialfor colony formation.

Example 6: Predictive Analysis of Regulatory Binding Sites within Exon 9

A bioinformatics analysis was performed to identify potential bindingmotifs for splicing regulators. In particular, algorithms that predictbinding sites for human splicing regulatory factors on the basis of thenucleotide sequence preferences obtained experimentally through variousapproaches (SpliceAid web tool, see http://www.introni.it/splicing.html)were used to identify potential binding sites for these factors withinthe sequence of exon 9. The whole human NUMB exon 9 sequence was used.The results are shown on FIG. 6 . The analysis revealed a potentialbinding site for SF1/BBP (Branchpoint Binding Protein) in the regionspanning +8 to +16 of NUMB exon 9 (GGCACTGACTC, SEQ ID NO: 300). Threemutants of the RG6-NUMB minigene were produced, as well as the wild typesequence (WT: GGCACTGACTC, SEQ ID NO: 301): Mut3, which is predicted todisrupt the SF1/BBP binding site (GGCACTGACTCCTC >GGCACTCACCTC, SEQ IDNO: 302); and Mut4 (GGCACTGACTCCTC >GGCACCGACTC, SEQ ID NO: 303) andMut1 (GGCACTGACTCCTC >GGTACTGACCTC, SEQ ID NO: 304), which are predictedto have a milder effect. The four minigenes (wild-type, Mut1, Mut3 andMut4) were co-transfected in HEK-293 cells at constant concentrationswith increasing concentrations of a SF1/BBP expression vector. 48 hoursafter transfection, RNA and protein samples were collected and thefraction of exon 9 inclusion (PSI) was quantified by RT-PCR andcapillary electrophoresis, while the levels of SF1/BBP overexpressionwere estimated by western blot using a specific antibody. While mutantMut1 slightly increased exon 9 skipping, Mut2 did not show any effect(data not shown). Overexpression of SF1/BBP showed a slight tendencytowards increased exon 9 inclusion for the three reporter minigenes, butthe effects were not quantitatively significant. It was thereforeconcluded that SF1/BBP (and in particular, inhibition of SF1/BBP bindingby action of the AONs of the invention) is unlikely to mediate theeffects of the target regions identified.

As the SF1/BBP motif (ACUNAC) is very similar to the consensus QKIbinding site (ACUAAY), which has been described to regulate NUMBalternative splicing in lung cancer (Zong et al., 2014), it was decidedto test the effect of QKI overexpression in the minigene reporterconstructs above. The results of this analysis are shown on FIGS. 7A and7B.

These results show that QKI overexpression (see FIG. 7A) led toincreased levels of exon 9 skipping in the wild-type reporter (FIG. 7B),which is consistent with previous results (Zong et al., 2014). Giventhat excess QKI causes NUMB exon 9 skipping, it is also unlikely thatQKI would be the transacting factor that promotes NUMB exon 9 inclusionas a result of the targeting of NUMB exon 9 by AON1. Indeed, thepresence of AON1 would likely compete for the binding site of QKI. Inother words, the fact that both AON1 and QKI promote exon skippingindicates that AON1 is unlikely to act by blocking QKI.

Example 7: AONs According to the Invention Reduce Exon 9 Inclusion andTumour Progression In Vivo

In order to demonstrate that the AONs of the invention can be effectivein vivo, 100 μl of AON1 at a concentration of 3,100 ng/μl in PBS (anapproximate concentration of 12 μg of AON1/gram of mouse weight) wereadministered intratracheally to a wild-type BL6 adult male mice. 78hours after the administration the animals were sacrificed and lung andliver samples were collected. RNA was isolated from the tissues andendogenous NUMB exon 9 PSI values were measured by RT-PCR. The resultsof this analysis are shown on FIG. 8 .

As shown, the administration of AON1 into the lung of healthy mice ledto a significant (Student's t-test, p. value <0.001) reduction in NUMBPSI values (FIG. 8 shows the results over 4 mice compared to a controlof 3 mice administered with random AON), while it did not havedetectable effects in the liver (data not shown).

This assay shows that despite the fact that the levels of exon 9inclusion in endogeneous NUMB transcripts in the lungs of healthy miceare wry low, AON1 was capable, when administered directly to the lung,of inducing a significant further increase in exon skipping. This datafurther indicates that a potent effect may be expected in tumours thatdisplay higher levels of NUMB exon 9 inclusion.

The inventors then went on to test whether AON1 could modulate NUMBalternative splicing in lung tumours. A genetic mouse model ofKRAS-G12V-driven non-small cell lung carcinoma (Guerra et al., 2003) wasused for this purpose. As illustrated on FIG. 9A, young adult mice weretreated with tamoxifen around 6 months of age to induce the activationof Cre, which by removing an engineered premature STOP codon before theoncogenic KRAS-G12V, leads to expression of the oncogenic version of theprotein. 6 to 8 months after the administration of tamoxifen, micestarted to develop lung adenomas. At that point mice were treated with asingle intratracheal administration of AON1, a random AON-RND (using anapproximate concentration of 12 μg of AON1-RND/gram of weight of themice) or a saline buffer and sacrificed 3 weeks after. Lung samples wereextracted and tumors were microdissected. Non-tumoral lung samples werealso collected. Samples were processed as above with healthy mice. Theresults of this analysis are shown on FIG. 9B (results obtained for 4tumors coming from 2 AON1 treated mice, 5 tumors coming from 3 micetreated with AON RND and 6 tumors coming from 2 saline treated mice).These results indicate that the levels of NUMB exon 9 inclusion intumors treated with control AONs or saline buffer were more than 3-foldhigher than in healthy tissue (compare PSI values of RND between FIGS. 8and 9B). The PSI values of non-tumoral tissue in KRAS-G12V-activatedmice were also higher and comparable to the tumors (data not shown),which may be explained by the existence of premalignant lesions inducedby oncogenic KRAS activation in the apparently non-tumoral tissue.Further, the administration of AON1 was able to reduce significantly thelevels of NUMB exon 9 inclusion in the treated tumors (see FIG. 9B). Nosignificant differences between administrating saline buffer or the RNDAON were observed, indicating that the control AON does not alter theratios of the NUMB isoforms.

Taken together, the results suggest that a single intratrachealadministration of AON1 is capable of significantly reducing NUMB exon 9inclusion in KRASG12V derived tumors, which suggests that it could alsohave an impact on the growth of these tumours. The change in NUMBsplicing was maintained at least during 3 weeks after the administration(see FIG. 9B), suggesting the possibility of long-term effects.

The effect of AON1 on tumour progression was also analysed, as explainedon FIG. 10A. The same lung cancer mouse model described above(KRAS-G12V) was used and 4-5 months after the tamoxifen administration,when the tumors were detectable by micro CT, mice were treated witheither AON1 or a random AON, RND. Weekly intranasal administrations of100 μl of AON (at a concentration of 3,100 ng/μl) were performed to eachmouse (regardless of their weight). Tumor growth was followed every twoweeks by micro CT.

The results of the follow-up on tumor growth are summarised in FIGS. 10Band 10C (3 mice treated with the RND AON and 2 with AON1). These datasuggest that the treatment of lung tumors in a KRAS-V12D mouse with AON1can have therapeutic effects for preventing tumor growth. 45 days afterstarting the treatment, the size of the AON1-treated tumors wassignificantly reduced while the control tumors continue to grow.

The effect of AONs according to the invention (in particular AON4-5, SEQID NO: 22) was analysed after intravenous injection to an adult (3months old) healthy mice. 100 μl of AON4-5 (SEQ ID NO:22), a random AON(RND) or an AON (SEQ ID NO: 25) targeting the 5′SS (SEQ ID NO: 27) at aconcentration of 3,100 ng/μl in PBS (an approximate concentration of 12μg of AON1/gram of mouse weight) were administered intravenously (byretro orbital sinus injection) to a wild-type BL6 adult male mice, asshown on FIG. 11A. Three once-daily injections were performed (i.e. aninjection was performed every 24 hours up to a total of 72 hours). 78hours after the last administration the animals were sacrificed and lungand liver samples were collected. RNA was isolated from the tissues andendogenous NUMB exon 9 PSI values were measured by RT-PCR. The resultsof this analysis are shown in FIGS. 11B and 11C (3 mice per condition),respectively for the liver and lung.

These data indicate that the effect associated with the AONs or theinvention may be dependent on the administration mode. For example, inorder to target the liver, intravenous administration may beappropriate, whereas for administration to the lung administration tothe respiratory system may be more appropriate.

Finally, the effect of AON1 (SEQ ID NO:12) and AON6-7 (SEQ ID NO: 24) ontumour growth in vivo was further tested using an orthotopic mouse modelof lung cancer, in which SCID-BEIGE mice of approximately 6 weeks areintratracheally inoculated with human A549 lung cancer cells that stablyexpress luciferase, as explained in FIG. 12A. The proper inoculation ofthe cells was verified by an in vivo analysis of the luminescent signal.Starting a month after the intratracheal inoculation, the tumour growthwas monitored weekly. The mice were anesthesised and 100 μl of asolution of luciferine in steril PBS (100 mg/Kg concentration) wasinjected intreaperitoneally; 10 minutes after injection the luminescentsignal was measured using an In Vivo Imaging System (IVIS) by PerkinElmer®. Once the tumoral mass reached a certain volume, the treatmentwas started. Mice were intranassally instillated with 100 μl of the AON(AON1 (SEQ ID NO: 12) or AON6-7 (SEQ ID NO: 24)) resuspended in sterilePBS at a concentration of 3,100 ng/μl. Tumor growth (fold change tumorvolume was monitored) and the probabilities of differential tumor growthbetween the groups was estimated using a log-linear random effectsmodel, as explained in Bates et al., 2015. The data shown on FIG. 12B(the continuous lines on FIG. 12B indicating averages of the respectivedata sets, the control data being identified with circles (n=15 mice),the AON1 data being identified by with triangles pointing down (n=11),and the AON6-7 data being identified with triangles pointing up (n=11))indicates that the AONs according to the invention reduce growth of lungcancer tumours in vivo (probability that the tumor growth is reduced ineach of the treatment groups compared to the control >0.99, where theprobability calculation is performed as explained above).

Example 8: AONs According to the Invention Reduce Exon 9 Inclusion inMultiple Types of Cancer

The effect of in vitro treatment of two different colon cancer celllines (HT29 and LS174T) with an AON according to the invention (AON4-5,SEQ ID NO: 22) was tested to show the wide applicability of the AONs ofthe invention. Populations of 250,000 HT29 cells and 250,000 LS147Tcells were cultured in 6-well plates 24 h prior to the transfection. Thecells were transfected using lipofectamine® RNAiMax from ThermoFisher,as explained below, with 100 nM final concentration of the indicatedAONs. Cells were collected 24 h after the transfection, RNA wasextracted and NUMB alternative splicing was analysed by RT-PCR andcapilar electrophoresis. The results are shown in FIG. 13 , whichdemonstrates that a significant (Students t-test, ***P-value <0.001)reduction in PSI of NUMB exon 9 can be observed compared to a control inwhich a random AON was transfected.

The effect of in vitro treatment on colony formation of the twodifferent colon cancer cell lines (HT29 and LS174T), a cervix cancercell line (HeLa), and a hepatocarcinoma cell line (HeG2) with anotherAON according to the invention (AON1, SEQ ID NO: 12) was then tested.The results of this analysis are shown in FIG. 14 . This data indicatesthat transfection with AON1 results in a significant (Student's t-test,*P-value <0.05) reduction in the number of colonies of LS147T (FIG.14A), HT29 (FIG. 14B), and HeLA (FIG. 14C). By contrast, transfectionwith AON1 results in a significant (Student's t-test, *P-value <0.05)increase in the number of colonies for HepG2 (FIG. 14D), in line withthe understanding that NUMB protein plays a protumoral role in HepG2(see e.g. Xie, C. et al., 2015).

The inventors then went on to investigate the effect of in vitrotreatment with AON1 (SEQ ID NO: 12) on colony formation and exon 9inclusion in a variety of cancer cell lines, compared to a random 21 ntlong oligonucleotide (2′-O-Methyl phosphorotiotated RNA of sequence SEQID NO: 357). All cell lines were transfected at least in technicaltriplicates. Different concentrations of AON were used, depending on thecell line (either 10 nM, 50 nM or 100 nM, as indicated in Table 1below). The number of colonies was normalised to the number of coloniesof the same cell line transfected with the same concentration of randomAON. Statistical significance was calculated using Student's t-test(***P-value <0.001, **P-value <0.01, *P-value <0.05). The results of thecolony counting experiments can be seen in FIG. 21 . The levels ofendogeneous NUMB transcript with or without exon 9 were quantified bycapillary electrophoresis in order to calculate the PSI in eachcondition, and a DPSI value was calculated by subtracting the PSI valuefor the control (random AON) from the PSI values for the cells treatedwith AON1. The results of this analysis can be found in Table 1 below.

TABLE 1 Numb exon 9 inclusion values for various cell lines treated withAON1 vs. control Cell line DPSI AON concentration [nM] A549 −0.47 100H1395 −0.41 10 H1666 −0.13 10 H1975 −0.34 50 KLC1 −0.20 100 KLC2 −0.14100 KLC3 −0.12 100 KLC4 −0.03 100 H226 −0.08 10 MCF7 −0.14 100 LS147−0.44 50 HT29 −0.64 5 Pc3 −0.40 5 DU145 −0.64 50 QGP1 NA 100 Capan2−0.81 50 HeLa NA 100 Saos2 −0.32 50 SK4SN NA 100 Huh7 −0.20 50 FTO-2B NA100 HepG2 −0.39 100

The results shown in FIG. 21 indicate that a statistically significantreduction in the number of colonies formed upon treatment with AON1(plain bars on FIG. 21 ) compared to a control oligonucleotide (Randomoligonucleotide, bars with dotted fill on FIG. 21 ) is obtained in fourdifferent human lung adenocarcinoma (LUAD) cell lines (A549, H1395,H1666, H1975) and four different mouse LUAD cell lines (96 KLC1, 97KLC2, MCGL KLC3, LS KLC4), at least one lung squamous cell carcinoma(LSCC) cell line (H226), at least one colon cancer cell line (HT29), twohuman prostate cancer cell lines (pc3, DU145), at least one pancreaticcancer cell line (QGP1), a human cervical cancer cell line (HeLa), andat least one hepatocarcinoma cell line (Huh7). A large but nonstatistically significant reduction of colony numbers was observed inbreast cancer (MCF7), neuroblastoma (SK4SN) and osteosarcoma (Saos2), aswell as pancreatic cancer cell line Capan2, colon cancer cell line LS147and rat hepatocarcinoma cell line FTO-2B. However, the lack ofstatistical significance in these cell lines appears to be attributableto large variability in the control conditions for these particularsamples, rather than a lack of effect of the AON of the invention.Further, the data shows that in HepG2, where NUMB is described as havinga protumoral activity, the treatment with AON1 has a proliferativeeffect. By contrast, in other hepatocarcinoma cell lines such as Huh7,where NUMB is anti-tumoral, the treatment with AON1 results in areduction of the number of colonies.

Therefore, the data show on FIG. 21 indicates that treatment an AONaccording to the invention (in particular AON1, SEQ ID NO: 12) canreduce cellular proliferation in a variety of cancers, and in particularin cancers for which the NUMB protein has an antitumoral role.

The effect of in vitro treatment with a different AON according to theinvention, in particular AON6-7 (SEQ ID NO: 24) on colony formation andexon 9 inclusion in a subset of the cancer cell lines tested above wasadditionally investigated, using a similar protocol. The results ofthese experiments are shown in FIG. 22 . The data of FIG. 22 indicatesthat a different AON according to the invention can also reduce cellproliferation in a variety of cancer cell lines. Indeed, significantresults (Student's t-test,*P-value <0.05) were obtained for H1666, pc3and QGP1 cell lines, and large but non-statistically significant resultswere obtained for H1395 and Capan2 cell lines (presumably due to highvariability in the control condition).

These results indicate that the AONs according to the invention may beuseful in the treatment of any cancer for which NUMB protein has anantitumoral role, such as e.g. lung adenocarcinoma, lung squamous cellcarcinoma, cervical cancer, breast cancer, colon cancer, prostatecancer, pancreatic cancer, osteosarcoma, neuroblastoma, and somehepatocarcinomas. Similar results are expected to be obtained with brainglioblastoma (e.g. T98G cell line), acute monocytic leukemia (e.g THP-1cell line), as well as kidney and colorectal cancers.

Example 9: AONs According to the Invention can be Modified

The experiments above were performed using 2′-O-methyl phosphothioate-modified antisense oligonucleotides of 21 nucleotides inlength. Similar effects on NUMB exon 9 splicing are expected to beobtained with AONs with lengths of between 18 and 25 nucleotides.

Further, similar effects on NUMB exon 9 splicing are expected to beobtained using AONs having different chemistries. For example, lockednucleid acids (LNAs, also known as ‘inaccessible RNA’) are expected tobe useful in the context of the invention. In particular, the followingLNAs (or AONs comprising such sequences) are expected to show an effecton NUMB exon 9 skipping: AGGCUGA (SEQ ID NO: 36), GUCAGUG (SEQ ID NO:37), CCAUUAG (SEQ ID NO: 38), CUGAGUC (SEQ ID NO: 39), AGUGCCA (SEQ IDNO: 40), AGGCUGAGUCAGUG (SEQ ID NO: 41), GUCAGUGCCAUUAG (SEQ ID NO: 42),or AGGCUGAGUCAGUGCCAUUAG (SEQ ID NO: 44). The AONs of sequenceAGGCUGAGUCAGUG (SEQ ID NO: 41) and GUCAGUGCCAUUAG (SEQ ID NO: 42) may beparticularly beneficial in promoting NUMB exon 9 skipping, and the AONof sequence AGGCUGAGUCAGUGCCAUUAG (SEQ ID NO: 44) may be especiallybeneficial in promoting NUMB exon 9 skipping. Any DNA version of theabove sequences (i.e. with Ts replacing Us) would also be expected toshow an effect on exon 9 skipping, and the sequences provided above areintended to encompass such DNA versions.

In order to verify this, the inventors tested whether an antisenseoligonucleotide with a locked nucleic acid (LNA) backbone chemistry andthe same sequence as AON1 (SEQ ID NO: 12) was capable of promoting NUMBexon 9 skipping in vitro. Technical triplicates of A549 lungadenocarcinoma cell cultures were transfected with 100 nM of: (1) an AONof sequence SEQ ID NO: 12 with a locked nucleic acid backbone; or (2) arandom LNA AON of sequence SEQ ID NO: 349. The levels of endogeneousNUMB transcript with or without exon 9 were quantified by capillaryelectrophoresis in order to calculate the PSI in each condition. Theresults of these experiments are shown in FIG. 15A, where it can be seenthat the modified AON is capable of significantly increasing NUMB exon 9skipping compared to a control (random AON) condition (Studentst-test,***P-value <0.001).

The inventors further verified that the modified AON is also capable ofreducing colony formation in cancer cells. In particular, the colonyformation capacity of A549 lung adenocarcinoma cell cultures transfectedwith 100 nM of: (1) an AON of sequence SEQ ID NO: 12 with a lockednucleic acid backbone; or (2) a random AON of sequence SEQ ID NO: 349,was tested. The results of these experiments are shown in FIG. 15B,where it can be seen that modified (LNA) AON is capable of significantlyreducing the colony formation capacity of lung adenocarcinoma cellscompared to a control (random AON) condition (Students t-test,*P-value<0.05).

Locked Nucleic Acids (LNAs) according to the invention may additionallyhave a higher affinity for their target sequence compared to anon-modified sequence of the same length. Therefore, LNAs according tothe invention may be efficient repressors of NUMB exon 9 inclusion evenat shorter lengths than e.g. the 21 nt AONs exemplified above. Thishypothesis was tested in Example 11 below.

Further, fully 2′-O-methyl modified AONs with a phosphorothioatebackbone are also expected to be useful in the medical treatments andmethods of the invention. In particular, an AON with the sequenceAGGCTGAGTCAGTGCCATTAG (SEQ ID NO: 43) in which all or some of thecytosine residues are methylated may be expected to show good results inNUMB exon 9 skipping and consequential therapeutic effects.

In order to test this, a 2′-O-methyl modified AON with aphosphorothioate backbone with the sequence of AON1 (i.e. an AON ofsequence SEQ ID NO: 12) was transfected in HEK-293T cells at aconcentration of 100 nM. A control condition in which a randomnucleotide sequence 2′-O-methyl modified AON with a phosphorothioatebackbone (SEQ ID NO: 357) was transfected in HEK-293T cells at aconcentration of 100 nM was tested in parallel. Both conditions weretested in triplicates. The levels of endogeneous NUMB transcript with orwithout exon 9 were quantified by capillary electrophoresis in order tocalculate the PSI in each condition. The results of these experimentsare shown on FIG. 16 , where it can be seen that the modified AON iscapable of significantly increasing NUMB exon 9 skipping compared to acontrol (random AON) condition (Students t-test,***P-value <0.001).

Together, these experiments show that AONs according to the inventionwith modified chemistries also have a beneficial effect on exon 9skipping and reduction in colony formation, and can be expected to havesimilar effects in vivo as demonstrated above for the non-modified AONs.

Example 10: AONs Targeting any Part of the Region of SEQ ID NO: 305Induce NUMB Exon 9 Skipping and Reduce Colony Formation Capacity of LungAdenocarcinoma Cells

One of the regions identified in Example 2, the region having thesequence of SEQ ID NO: 305 (targeted by AON1, AON2, AON1-2, AON2-3; i.e.SEQ ID NO: 12, 13, 19 and 20) was further studied in order to identifywhether targeting specific sub-regions of this region of NUMB exon 9leads to stronger effects on NUMB exon 9 skipping and colony formation.

In particular, very low concentrations (10 nM) of 2′O-Methylphosphorothioated RNA with the sequence of each of AON1 (SEQ ID NO: 12),AON1-2 (SEQ ID NO: 19), AON2 (SEQ ID NO: 13), AON2-3 (SEQ ID NO: 20),and a random 2′O-Methyl phosphorothioated RNA AON (SEQ ID NO: 357) weretransfected in A549 lung adenocarcinoma cells. The levels of NUMBtranscript with or without exon 9 were quantified by capillaryelectrophoresis in order to calculate the endogeneous NUMB exon 9 PSI ineach condition, and the number of colonies was counted in eachcondition.

The results of these experiments are shown in FIGS. 17A (endogeneousNUMB exon 9 PSI) and 17B (colony formation), where all statistics wereobtained using Student's t-test (***P-value <0.001). These results showthat targeting any part of the region of sequence SEQ ID NO: 305 in exon9 of NUMB results in significant induction of exon 9 skipping andreduction of colony formation capacity, since even at low concentrations(where any differences in potency between the different sub-areas of theregion would be expected to appear more prominently) all of thesub-regions are associated with similar levels of potency. As such, thedata supports the hypothesis that targeting any part of the entireregion of SEQ ID NO: 305 is associated with potent inhibition of exon 9inclusion.

Example 11: Short AONs Targeting Sub-Regions of Region 1 Induce NUMBExon 9 Skipping

In order to further characterise sub-regions within the region of SEQ IDNO: 305, and in particular within Region 1 (SEQ ID NO: 12), AONs withshorter lengths than AON1 were then produced. These were produced with alocked nucleic acid (LNA) backbone chemistry in order to simultaneouslytest the hypothesis that such modified AONs may be expected to have highaffinity for their target even when using shorter lengths of AONs.

In particular, the inventors designed LNA AONs according to theinvention, targeting the region defined by SEQ ID NO: 305 in NUMB exon9, with lengths of 7 nucleotides, 14 and 21 nucleotides. These weretransfected at a concentration of 100 nM in HEK-293T cells. A controlAON (2′-O-methyl modified AON with a phosphorothioate backbone with arandom sequence of SEQ ID NO: 357) was also transfected at aconcentration of 100 nM in HEK-293T cells. The levels of NUMB transcriptwith or without exon 9 were quantified by capillary electrophoresis inorder to calculate the endogeneous NUMB exon 9 PSI in each condition.

The following five 7-nucleotide LNA AONS were tested: LNA1 is an AON ofsequence SEQ ID NO: 344, which targets SEQ ID NO: 59; LNA1-2 is an AONof sequence SEQ ID NO: 345, which targets SEQ ID NO: 56; LNA2 is an AONof sequence SEQ ID NO: 346, which targets SEQ ID NO: 52; LNA2-3 is anAON of sequence SEQ ID NO: 347, which targets SEQ ID NO: 49; and LNA3 isan AON of sequence SEQ ID NO: 348, which targets SEQ ID NO: 45. All ofthe 7 nt AONs target regions also targeted by AON1 (SEQ ID NO: 12), i.e.Region 1 of Homo sapiens NUMB exon 9 (SEQ ID NO: 44).

The following 14-nucleotide LNA AONS were tested: LONG1 is an AON ofsequence SEQ ID NO: 339, which targets SEQ ID NO: 341; LONG2 is an AONof sequence SEQ ID NO: 340, which targets SEQ ID NO: 342. Both LONG1 andLONG2 target regions also targeted by AON1 (SEQ ID NO: 12), i.e. Region1 of Homo sapiens NUMB exon 9 (SEQ ID NO: 44).

Additionally, a 21 nucleotides long LNA AON with the sequence of AON1(SEQ ID NO: 12) was also tested (referred to as “SUPERLONG” in FIG.18A).

The results of these experiments can be seen in FIG. 18A, where allstatistics are the result of Student's t-test ((***P-value <0.001). Ascan be seen in FIG. 18A, all five of the short LNAs (7 nt long) resultedin similar and significant levels of exon 9 exclusion, regardless of thesub-region of the region of SEQ ID NO:12 that was targeted. Further,both of the 14 nt LNA AONs also resulted in significant levels of exon 9skipping, which were higher than that observed using the 7 nt long AONs.LONG2 appeared to be slightly more potent than LONG1. The LNA AON of 21nt shows the strongest effect of all constructs tested.

The same experiment was repeated with higher doses of each AON (1,000nM), and the results can be seen in FIG. 18B (where SL LNA1 is theSUPERLONG LNA AON of FIG. 18A). As can be seen in FIG. 18B, increasingthe dose results in slightly lower levers of exon 9 inclusion, but thesame patterns as in FIG. 18A can be observed. This may indicate thateither the lower concentration of 100 nM already almost saturated thesystem, or that the two concentrations are associated with differenttransfection efficacies.

The data in FIGS. 18A and 18B indicates that any sub-region of Region 1of Homo sapeins exon 9 (SEQ ID NO: 44) can be efficiently targeted byAONs as short as 7 nt, even at concentrations as low as 100 nM, at leastwhen using LNA modified AONs.

The slight difference between LONG1 (LNA AON of SEQ ID NO: 339) andLONG2 (LNA AON of SEQ ID NO: 340) was further investigated bytransfecting the constructs in A549 cells at a lower concentration ofnM. A comparative random LNA AON of 14 nt (SEQ ID NO: 343) was tested inparallel. The levels of NUMB transcript with or without exon 9 werequantified by capillary electrophoresis in order to calculate theendogeneous NUMB exon 9 PSI in each condition, and the number ofcolonies was counted in each condition.

The results of these experiments are shown in FIGS. 19A (endogeneousNUMB exon 9 PSI) and 19B (colony formation), where all statistics wereobtained using Student's t-test (***P-value <0.001, *P-value <0.05).This data shows that the slightly stronger effect on exon 9 inclusionseen with LONG2 compared to LONG1 in FIGS. 18A and 18B is still apparentat this lower concentration, although both constructs resulted insignificant inhibition of exon 9 inclusion compared to the randomcontrol. The difference between LONG1 and LONG2 was less visible withrespect to colony formation, although LONG2 appears to reduce the numberof colonies in a more consistent fashion than LONG1. Together, theseresults indicate that the first 14 nt sequence of Region 1 (SEQ ID NO:12) may contain regulatory elements that are particularly important ininducing exon 9 skipping. However, the data in FIGS. 18A and 18Bindicates that targeting Region 1 outside of this first 14 nt sequence(see the results obtained with LNA1 and LNA1-2) still results insignificant inhibition of exon 9 inclusion.

FIG. 20 summarises the AONs that were tested in the experiments ofExamples 10 and 11, in which the naming of any AON also tested inExample 2 (as shown in FIG. 2 ), is consistent with the naming used inExample 2/FIG. 2 .

Materials and Methods Study of NUMB Exon 9 Inclusion in DifferentTumours:

The analysis of NUMB exon 9 PSI distribution in cancer samples wasderived from (Sebestyen et al. 2015). NUMB exon 9 PSI was comparedacross 11 different tumor types using only paired tumor/non-tumorsamples from the TCGA consortium. Mann Whitney U test was used to assessdifferences in PSI distribution between of healthy and tumor samples;correction of multiple testing was performed with Benjamini-Hochberg.

Scanning of NUMB Exon 9 with AONS:

2′-O-methyl phosphothioate-modified antisense oligonucleotides of 21nucleotides in length and reverse-complementary to NUMB exon 9 sequencewere designed, with sequences SEQ ID NO: 12 to 24. The compounds weresynthesised by Sigma-Aldrich. Two batches of AONs were ordered, one of 8mg and one of 16 mg. AONs were ordered dry, resuspended in PBS andstored at −20° C.

A stably transformed HEK-293T cell line expressing a reporter NUMB exon9 minigene (RG6-NUMB, (Bechara et al., 2013; Hernandez et al., 2016))was transfected with 100 nM of individual AONs 24 hours aftertransfection, RNA was isolated, retro-transcribed and amplified withprimers specific for detecting NUMB exon 9 inclusion/skipping in theminigene. The results of NUMB exon 9 inclusion (PSI) was calculated forthree technical replicates for each AON.

Cell Culture and Transfection:

All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and antibiotics(50 units/ml penicillin and 50 ml/ml streptomycin).

Cell transfection, RNA extraction and processing (includingreverse-transcription, PCR amplification and isoform quantification bycapillary electrophoresis) were performed as in (Hernandez et al. 2016).PSI quantification was performed either by capillary electrophoresis(Hernandez et al. 2016) or by electrophoretic separation of the PCRproducts in 6% acrylamide gels. In the latter case, SyberGreen was usedfor band detection, images were acquired with Geldoc (Biorad) and theband quantification was performed with FIJI software (Schindelin et al.,2012).

For clonogenic assays, 20,000 mice cells were cultured in 6-well plates(2000 A549 cells) and transfected using Lipofectamine RNAiMAX(Invitrogen) with 100 nM of AONs, following the manufacturer'sinstructions. Cells were maintained in culture for at least 7 days, thenmedia was removed and cells were fixed with 1 ml methanol and stainedwith crystal violet overnight. Afterwards, the wells were washed withwater and left to dry. Colony quantification was performed using FIJIsoftware and a custom-made R script.

Prediction of Splicing Factor Binding Sites:

SpliceAid2 web tool was used for predicting splicing factor bindingsites across NUMB exon 9 (Piva et al., 2012;(http://193.206.120.249/splicing_tissue.html).

Mice Experiments:

Wild-type BL6 male mice, older than 12 weeks were used for testing theeffect of AONs in vivo in healthy lungs. AONs were resuspended at aconcentration of 3,100 ng/μl, and a volume of 100 μl was intratracheallyadministered to the mice (regardless of their weight). 3 days after theadministration, mice were sacrificed and samples were collected. Tissuewas disaggregated using glass-beads (Glass-beads acid-washed, 425 to 600μm, Sigma) and a beadbeater (Mini-Beadbeater, Biospec Products), thenresuspended in homogenisation buffer from Maxwell 16 LEV simple RNAtissue kit (Promega). After tissue disruption, RNA was isolated from thesamples using Maxwell robot following Maxwell 16 LEV simple RNA tissuekit manufacturer's instructions.

RERTert, K-rasLSLG12V mice (Guerra et al. 2003) were used for testingthe effect of AONs on tumors. 22 week old mice were injectedintraperitoneally with tamoxifen (1 mg/mice) to induce tumor formation.Once tumors could be detected by micro-CT (6 to 8 months) a singleadministration of AON was performed (3,100 ng/μl AONs in saline buffer,100 μl/mice) intratracheally. After 3 weeks, mice were sacrificed andtissue samples were collected. Lung tumors were micro-dissected andhealthy and tumor tissues were processed in parallel as explained forhealthy mice tissue (see above).

For experiments relating to tumor progression, the same mouse model(RERTert, K-rasLSLG12V mice) and protocol was used. Once mice displayedtumors, they were administered the same dose of AON once a week for twomonths via intranasal administration. Tumor growth was followed bymicro-CT every other week for two months.

For the orthotopic mouse model, SCID-Beige mice of at least 6 weeks ofage where deeply anesthesised and inoculated, intratracheally with1,500,000 A549 cells with a plasmid encoding luciferase. One month afterthe inoculation the mice were monitored weekly for the detection ofluminescents signal: a read out of the presence of the cancerous cells.Mice were anestesised and injected, intraperitoneally with 100 μl ofluciferine (100 mg/kg concentration, resuspended in PBS), and 10 minuteslater, the luminescent signal was quantified with the In Vivo ImagingSystem (IVIS) by Perkin Elmer®. Once the tumors were determined to havegrown to a given size, mice were treated once a week with theoligonucleotide of the invention for two months. 100 μl of 3,100 ng/μl(resuspended in sterile PBS) of AON were delivered to the miceintranasally.

REFERENCES

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Sequences:

In the antisense oligonucleotide sequences provided, any U can be readas a T in alternative embodiments which are explicitly intended to beencompassed within the scope of the claimed invention.

SEQ ID NO: Sequence Type Sequence   1 Mouse NUMB exon 9ctaatggcac tgactcagcc tcccatgtgc ttactgctaa(NM_001136075.2-Numb mRNA transcript gccagccaata ctgctctagc acacgtagcavariant 1-1115 . . . 1261) atgcctgtcc gtgaaaccaa cccctgggcc catgtccctgatgctgctaa caaggaaatt gcagccatac atccgg   2 Human NUMB exon 9ctaatggcac tgactcagcc ttccatgtgc ttgctaagcc(NM_001005743-Numb mRNA transcriptagcccatact gctctagcac ccgtagcaat gcctgtgcgt variant 1-1417 . . . 1560)gaaaccaacc cttgggccca tgcccctgat gctgctaacaa ggaaattgca gccacatgtt cgg 44 Region 1 of Homo sapiens Numb exon 9 CUAAUGGCACUGACUCAGCCU(targeted by AON1-SEQ ID NO: 12)   3Region 2 of Homo sapiens Numb exon 9 CCUUCCAUGUGCUUGCUAAGC(targeted by AON2-SEQ ID NO: 13)   4Region 3 of Homo sapiens Numb exon 9 CAGCCCAUACUGCUCUAGCAC(targeted by AON3-SEQ ID NO: 14)   5Region 4 of Homo sapiens Numb exon 9 CCGUAGCAAUGCCUGUGCGUG(targeted by AON4-SEQ ID NO: 15)   6Region 5 of Homo sapiens Numb exon 9 AAACCAACCCUUGGGCCCAUG(targeted by AON5-SEQ ID NO: 16)   7Region 6 of Homo sapiens Numb exon 9 CCCCUGAUGCUGCUAACAAGG(targeted by AON6-SEQ ID NO: 17)   8Region 7 of Homo sapiens Numb exon 9 AAAUUGCAGCCACAUGUUCGG(targeted by AON7-SEQ ID NO: 18)   9Region 1+2 of Homo sapiens Numb exon 9 CUAAUGGCACUGACUCAGCCUUCCAUG(targeted by AON1-SEQ ID NO: 12; AON2- UGCUUGCUAAGCSEQ ID NO: 13; and AON1-2-SEQ ID NO: 19)  10Region 4+5 of Homo sapiens Numb exon 9 CCGUAGCAAUGCCUGUGCGUGAAACCA(targeted by AON4-SEQ ID NO: 15; AON5- ACCCUUGGGCCCAUGSEQ ID NO: 16; and AON4-5-SEQ ID NO: 22)  11Region 6+7 of Homo sapiens Numb exon 9 CCCCUGAUGCUGCUAACAAGGAAAUUGC(targeted by AON6-SEQ ID NO: 17; AON7- AGCCACAUGUUCGGSEQ ID NO: 18 and AON6-7-SEQ ID NO: 24)  12 Antisense oligonucleotideAGGCUGAGUCAGUGCCAUUAG targeting Region 1 ofHomo sapiens Numb exon 9 (AON1)  13 Antisense oligonucleotideGCUUAGCAAGCACAUGGAAGG targeting Region 2 ofHomo sapiens Numb exon 9 (AON2)  14 Antisense oligonucleotideGUGCUAGAGCAGUAUGGGCUG targeting Region 3 ofHomo sapiens Numb exon 9 (AON3)  15 Antisense oligonucleotideCACGCACAGGCAUUGCUACGG targeting Region 4 ofHomo sapiens Numb exon 9 (AON4)  16 Antisense oligonucleotideCAUGGGCCCAAGGGUUGGUUU targeting Region 5 ofHomo sapiens Numb exon 9 (AON5)  17 Antisense oligonucleotideCCUUGUUAGCAGCAUCAGGGG targeting Region 6 ofHomo sapiens Numb exon 9 (AON6)  18 Antisense oligonucleotideCCGAACAUGUGGCUGCAAUUU targeting Region 7 ofHomo sapiens Numb exon 9 (AON7)  19 Antisense oligonucleotideCACAUGGAAGGCUGAGUCAGU targeting Region 1-2of Homo sapiens Numb exon 9 (AON1-2)  20 Antisense oligonucleotideAGUAUGGGCUGGCUUAGCAAG targeting Region 2-3of Homo sapiens Numb exon 9 (AON2-3)  21 Antisense oligonucleotideCAUUGCUACGGGUGCUAGAGC targeting Region 3-4of Homo sapiens Numb exon 9 (AON3-4)  22 Antisense oligonucleotideAGGGUUGGUUUCACGCACAGG targeting Region 4-5of Homo sapiens Numb exon 9 (AON4-5)  23 Antisense oligonucleotideAGCAUCAGGGGCAUGGGCCCA targeting Region 5-6of Homo sapiens Numb exon 9 (AON5-6)  24 Antisense oligonucleotideGGCUGCAAUUUCCUUGUUAGC targeting Region 6-7of Homo sapiens Numb exon 9 (AON6-7)  25 Antisense oligonucleotideAUCUGUGGCCACCUUACCCGA targeting the 5′ splice site of Numb exon 9  26Antisense oligonucleotide UUAGCUACAACGGGAGCAGAC targeting the 3′ splicesite of Homo sapiens Numb exon 9  27 Homo sapiens Numb exon 9 sequenceUCGGGUAAGGUGGCCACAGAU targeted by antisense oligonucleotideof SEQ. ID NO: 25  28 Homo sapiens Numb exon 9 sequenceGTCTGCTCCCGTTG TAGCTAA targeted by antisense oligonucleotideof SEQ. ID NO: 26  29 Antisense oligonucleotide CAACAUCAAUGGAGUUAAUUCAUtargeting the branching point of Homo sapiens Numb exon 9  30Region of Homo sapiens ACUGACUCAGCCUUCCAUGUG Numb exon 9 targetedby SEQ.ID NO: 19 (AON1-2)  31 Region of Homo sapiensCCUGUGCGUGAAACCAACCCU Numb exon 9 targeted by SEQ.ID NO: 22 (AON4-5)  32Region of Homo sapiens GCUAACAAGGAAAUUGCAGCC Numb exon 9 targetedby SEQ.ID NO: 24 (AON6-7)  33 Region of Homo sapiensCUUGCUAAGCCAGCCCAUACU Numb exon 9 targeted by SEQ.ID NO: 20 (AON2-3)  34Region of Homo sapiens GCUCUAGCACCCGUAGCAAUG Numb exon 9 targetedby SEQ.ID NO: 21 (AON3-4)  35 Region of Homo sapiensUGGGCCCAUGCCCCUGAUGCU Numb exon 9 targeted by SEQ.ID NO: 23 (AON5-6)  36Antisense oligonucleotide AGGCUGA targeting Region 1 of Homo sapiensNumb exon 9  37 Antisense oligonucleotide GUCAGUG targeting Region 1 ofHomo sapiens Numb exon 9  38 Antisense oligonucleotide CCAUUAGtargeting Region 1 of Homo sapiens Numb exon 9  39Antisense oligonucleotide CUGAGUC targeting Region 1-2 of Homo sapiensNumb exon 9  40 Antisense oligonucleotide AGUGCCA targeting Region 2-3of Homo sapiens Numb exon 9  41 Antisense oligonucleotide AGGCUGAGUCAGUGtargeting Region 1 of Homo sapiens Numb exon 9  42Antisense oligonucleotide GUCAGUGCCAUUAG targeting Region 1 ofHomo sapiens Numb exon 9  43 Antisense oligonucleotideAGGCMeUGAGUCMeAGUGCMecMeAUUAG targeting Region 1 of Homo sapiensNumb exon 9  45-77 Target regions in Region 1+2 CUAAUGG of Homo sapiensUAAUGGC Numb exon 9 AAUGGCA AUGGCAC UGGCACU GGCACUG GCACUGA CACUGACACUGACU CUGACUC UGACUCA GACUCAG ACUCAGC CUCAGCC UCAGCCU CAGCCUU AGCCUUCGCCUUCC CCUUCCA CUUCCAU UUCCAUG UCCAUGU CCAUGUG CAUGUGC AUGUGCU UGUGCUUGUGCUUG UGCUUGC GCUUGCU CUUGCUA UUGCUAA UGCUAAG GCUAAGC  78-104Target regions in Region 1+2 CUAAUGGCACUGA of Homo sapiens UAAUGGCACUGACNumb exon 9 AAUGGCACUGACU AUGGCACUGACUC UGGCACUGACUCA GGCACUGACUCAGGCACUGACUCAGC CACUGACUCAGCC ACUGACUCAGCCU CUGACUCAGCCUU UGACUCAGCCUUCGACUCAGCCUUCC ACUCAGCCUUCCA CUCAGCCUUCCAU UCAGCCUUCCAUG CAGCCUUCCAUGUAGCCUUCCAUGUG GCCUUCCAUGUGC CCUUCCAUGUGCU CUUCCAUGUGCUU UUCCAUGUGCUUGUCCAUGUGCUUGC CCAUGUGCUUGCU CAUGUGCUUGCUA AUGUGCUUGCUAA UGUGCUUGCUAAGGUGCUUGCUAAGC 105-123 Target regions in Region 1+2 CUAAUGGCACUGACUCAGCCUof Homo sapiens UAAUGGCACUGACUCAGCCUU Numb exon 9 AAUGGCACUGACUCAGCCUUCAUGGCACUGACUCAGCCUUCC UGGCACUGACUCAGCCUUCCA GGCACUGACUCAGCCUUCCAUGCACUGACUCAGCCUUCCAUG CACUGACUCAGCCUUCCAUGU ACUGACUCAGCCUUCCAUGUGCUGACUCAGCCUUCCAUGUGC UGACUCAGCCUUCCAUGUGCU GACUCAGCCUUCCAUGUGCUUACUCAGCCUUCCAUGUGCUUG CUCAGCCUUCCAUGUGCUUGC UCAGCCUUCCAUGUGCUUGCUCAGCCUUCCAUGUGCUUGCUA AGCCUUCCAUGUGCUUGCUAA GCCUUCCAUGUGCUUGCUAAGCCUUCCAUGUGCUUGCUAAGC 124-159 Target regions in Region 4+5 CCGUAGCof Homo sapiens CGUAGCA Numb exon 9 GUAGCAA UAGCAAU AGCAAUG GCAAUGCCAAUGCC AAUGCCU AUGCCUG UGCCUGU GCCUGUG CCUGUGC CUGUGCG UGUGCGU GUGCGUGUGCGUGA GCGUGAA CGUGAAA GUGAAAC UGAAACC GAAACCA AAACCAA AACCAAC ACCAACCCCAACCC CAACCCU AACCCUU ACCCUUG CCCUUGG CCUUGGG CUUGGGC UUGGGCC UGGGCCCGGGCCCA GGCCCAU GCCCAUG 160-189 Target regions in Region 4+5CCGUAGCAAUGCC of Homo sapiens CGUAGCAAUGCCU Numb exon 9 GUAGCAAUGCCUGUAGCAAUGCCUGU AGCAAUGCCUGUG GCAAUGCCUGUGC CAAUGCCUGUGCG AAUGCCUGUGCGUAUGCCUGUGCGUG UGCCUGUGCGUGA GCCUGUGCGUGAA CCUGUGCGUGAAA CUGUGCGUGAAACUGUGCGUGAAACC GUGCGUGAAACCA UGCGUGAAACCAA GCGUGAAACCAAC CGUGAAACCAACCGUGAAACCAACCC UGAAACCAACCCU GAAACCAACCCUU AAACCAACCCUUG AACCAACCCUUGGACCAACCCUUGGG CCAACCCUUGGGC CAACCCUUGGGCC AACCCUUGGGCCC ACCCUUGGGCCCACCCUUGGGCCCAU CCUUGGGCCCAUG 190-211 Target regions in Region 4+5CCGUAGCAAUGCCUGUGCGUG of Homo sapiens CGUAGCAAUGCCUGUGCGUGA Numb exon 9GUAGCAAUGCCUGUGCGUGAA UAGCAAUGCCUGUGCGUGAAA AGCAAUGCCUGUGCGUGAAACGCAAUGCCUGUGCGUGAAACC CAAUGCCUGUGCGUGAAACCA AAUGCCUGUGCGUGAAACCAAAUGCCUGUGCGUGAAACCAAC UGCCUGUGCGUGAAACCAACC GCCUGUGCGUGAAACCAACCCCCUGUGCGUGAAACCAACCCU CUGUGCGUGAAACCAACCCUU UGUGCGUGAAACCAACCCUUGGUGCGUGAAACCAACCCUUGG UGCGUGAAACCAACCCUUGGG GCGUGAAACCAACCCUUGGGCCGUGAAACCAACCCUUGGGCC GUGAAACCAACCCUUGGGCCC UGAAACCAACCCUUGGGCCCAGAAACCAACCCUUGGGCCCAU AAACCAACCCUUGGGCCCAUG 212-247Target regions in Region 6+7 CCCCUGA of Homo sapiens CCCUGAU Numb exon 9CCUGAUG CUGAUGC UGAUGCU GAUGCUG AUGCUGC UGCUGCU GCUGCUA CUGCUAA UGCUAACGCUAACA CUAACAA UAACAAG AACAAGG ACAAGGA CAAGGAA AAGGAAA AGGAAAU GGAAAUUGAAAUUG AAAUUGC AAUUGCA AUUGCAG UUGCAGC UGCAGCC GCAGCCA CAGCCAC AGCCACAGCCACAU CCACAUG CACAUGU ACAUGUU CAUGUUC AUGUUCG UGUUCGG 248-277Target regions in Region 6+7 CCCCUGAUGCUGC of Homo sapiens CCCUGAUGCUGCUNumb exon 9 CCUGAUGCUGCUA CUGAUGCUGCUAA UGAUGCUGCUAAC GAUGCUGCUAACAAUGCUGCUAACAA UGCUGCUAACAAG GCUGCUAACAAGG CUGCUAACAAGGA UGCUAACAAGGAAGCUAACAAGGAAA CUAACAAGGAAAU UAACAAGGAAAUU AACAAGGAAAUUG ACAAGGAAAUUGCCAAGGAAAUUGCA AAGGAAAUUGCAG AGGAAAUUGCAGC GGAAAUUGCAGCC GAAAUUGCAGCCAAAAUUGCAGCCAC AAUUGCAGCCACA AUUGCAGCCACAU UUGCAGCCACAUG UGCAGCCACAUGUGCAGCCACAUGUU CAGCCACAUGUUC AGCCACAUGUUCG GCCACAUGUUCGG 278-299Target regions in Region 6+7 CCCCUGAUGCUGCUAACAAGG of Homo sapiensCCCUGAUGCUGCUAACAAGGA Numb exon 9 CCUGAUGCUGCUAACAAGGAACUGAUGCUGCUAACAAGGAAA UGAUGCUGCUAACAAGGAAAU GAUGCUGCUAACAAGGAAAUUAUGCUGCUAACAAGGAAAUUG UGCUGCUAACAAGGAAAUUGC GCUGCUAACAAGGAAAUUGCACUGCUAACAAGGAAAUUGCAG UGCUAACAAGGAAAUUGCAGC GCUAACAAGGAAAUUGCAGCCCUAACAAGGAAAUUGCAGCCA UAACAAGGAAAUUGCAGCCAC AACAAGGAAAUUGCAGCCACAACAAGGAAAUUGCAGCCACAU CAAGGAAAUUGCAGCCACAUG AAGGAAAUUGCAGCCACAUGUAGGAAAUUGCAGCCACAUGUU GGAAAUUGCAGCCACAUGUUC GAAAUUGCAGCCACAUGUUCGAAAUUGCAGCCACAUGUUCGG 300 Potential binding site of GGCACTGACTCSF 1/BBP in Numb exon 9 301 RG6-NUMB minigene WT GGCACTGACTC 302RG6-NUMB minigene Mut3 GGCACTCACCTC 303 RG6-NUMB minigene Mut 4GGCACCGACTC 304 RG6-NUMB minigene Mut 1 GGTACTGACCTC 305Region 1+2+ 2−3 of Homo sapiens CUAAUGGCACUGACUCAGCCUUCCAUGNumb exon 9 (targeted by UGCUUGCUAAGCCAGCCCAUACUby AON1-SEQ ID NO: 12; AON2 SEQ ID NO: 13; AON1-2- SEQ ID NO: 19; andAON2-3-SEQ ID NO: 20) 306-316 Target regions in Region 1+2+2−3 ofSEQ ID NO: 45-77 Homo sapiens CUAAGCC Numb exon 9 UAAGCCA AAGCCAGAGCCAGC GCCAGCC CCAGCCC CAGCCCA AGCCCAU GCCCAUA CCCAUAC CCAUACU 317-327Target regions in Region 1+2+2−3 SEQ ID NO: 78-104 of Homo sapiensUGCUUGCUAAGCC Numb exon 9 GCUUGCUAAGCCA CUUGCUAAGCCAG UUGCUAAGCCAGCUGCUAAGCCAGCC GCUAAGCCAGCCC CUAAGCCAGCCCA UAAGCCAGCCCAU AAGCCAGCCCAUAAGCCAGCCCAUAC GCCAGCCCAUACU 328-338 Target regions in Region 1+2+2−3SEQ ID NO: 105-123 of Homo sapiens CUUCCAUGUGCUUGCUAAGCC Numb exon 9UUCCAUGUGCUUGCUAAGCCA UCCAUGUGCUUGCUAAGCCAG CCAUGUGCUUGCUAAGCCAGCCAUGUGCUUGCUAAGCCAGCC AUGUGCUUGCUAAGCCAGCCC UGUGCUUGCUAAGCCAGCCCAGUGCUUGCUAAGCCAGCCCAU UGCUUGCUAAGCCAGCCCAUA GCUUGCUAAGCCAGCCCAUACCUUGCUAAGCCAGCCCAUACU 339 Antisense oligonucleotide AGGCUGAGUCAGUGtargeting Region 1 of Homo sapiens Numb exon 9-14 nt long,targeting second part of region defined by SEQ ID NO: 44 340Antisense oligonucleotide GUCAGUGCCAUUAG targeting Region 1 ofHomo sapiens Numb exon 9-14 nt long, targeting first part ofregion defined by SEQ ID NO: 44 341 Homo sapiens CACUGACUCAGCCUNumb exon 9 sequence targeted by antisense oligonucleotideof SEQ ID NO: 339 342 Homo sapiens CUAAUGGCACUGACNumb exon 9 sequence targeted by antisense oligonucleotideof SEQ ID NO: 340 343 Random oligonucleotide-14 nt long AAACCGCGCGUACG344 Antisense oligonucleotide AGGCUGA targeting SEQ IDNO: 59 in Region 1 of Homo sapiens Numb exon 9-7 nt long 345Antisense oligonucleotide CUGAGUC targeting SEQ IDNO: 56 in Region 1 of Homo sapiens Numb exon 9-7 nt long 346Antisense oligonucleotide GUCAGUG targeting SEQ ID NO:52 in Region 1 of Homo sapiens Numb exon 9- 7 nt long 347Antisense oligonucleotide AGUGCCA targeting SEQ IDNO: 49 in Region 1 of Homo sapiens Numb exon 9-7 nt long 348Antisense oligonucleotide CCAUUAG targeting SEQ ID NO:45 in Region 1 of Homo sapiens Numb exon 9-7 nt long 349Random oligonucleotide-21 nt long AACCGCGCGUACGAAACCGUC 350Antisense oligonucleotide CAAGCACAUGGAAGGCUGAGU targeting SEQ ID NO:117 in Region 1+2+2−3 of Homo sapiens Numb exon 9-21 nt long 351Antisense oligonucleotide UUAGCAAGCACAUGGAAGGCU targeting SEQ ID NO:121 in Region 1+2+2−3 of Homo sapiens Numb exon 9-21 nt long 352Antisense oligonucleotide GCUGGCUUAGCAAGCACAUGG targeting SEQ ID NO:331 in Region 1+2+2−3 of Homo sapiens Numb exon 9-21 nt long 353Antisense oligonucleotide GAGCAGUAUGGGCUGGCUUAG targeting SEQ ID NO:in Region 2+3 of Homo sapiens Numb exon 9- 21 nt long 354Region of Homo sapiens CUAAGCCAGCCCAUACUGCUC Numb exon 9 targetedby SEQ ID NO: 353 355 Antisense oligonucleotide GCUAGAGCAGUAUGGGCUGGCtargeting a in Region 2+3 of Homo sapiens Numb exon 9-21 nt long 356Region of Homo sapiens GCCAGCCCAUACUGCUCUAGC Numb exon 9 targetedby SEQ ID NO: 355 357 21 nt random oligonucleotide,UGAUUCGUGCGGCGCGUAUAU 2+40 -O-Methyl Phosphorothioated

1-15. (canceled)
 16. An antisense oligonucleotide for reducing inclusionof NUMB exon 9 in a population of mature NUMB transcripts, the antisenseoligonucleotide comprising a sequence of at least 7 nucleotides that iscomplementary to a target region of a NUMB transcript within exon 9comprising SEQ ID NO: 2 or a homologue thereof.
 17. The antisenseoligonucleotide of claim 16, wherein the target region is comprisedwithin the sequence CUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGCCAGCCCAUACU(SEQ ID NO: 305), CCGUAGCAAUGCCUGUGCGUGAAACCAACCCUUGGGCCCAUG (SEQ ID NO:10), or CCCCUGAUGCUGCUAACAAGGAAAUUGCAGCCACAUGUUCGG (SEQ ID NO: 11), or ahomologue thereof.
 18. The antisense oligonucleotide of claim 16,wherein the target region is comprised within the sequenceCUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGC (SEQ ID NO: 9),CUAAUGGCACUGACUCAGCCU (SEQ. ID NO: 44), CCUGUGCGUGAAACCAACCCU (SEQ IDNO: 31), GCUAACAAGGAAAUUGCAGCC (SEQ ID NO: 32) or UGGGCCCAUGCCCCUGAUGCU(SEQ ID NO: 35), or a homologue thereof.
 19. The antisenseoligonucleotide of claim 16, consisting essentially of a nucleotidesequence complementary to a contiguous nucleotide sequence of a NUMBtranscript.
 20. The antisense oligonucleotide of claim 16, wherein theantisense oligonucleotide comprises a nucleotide sequence that iscomplementary to a target region comprising between 7 and 25 nucleotidesof NUMB exon 9; or is complementary to a target region of at least 13nucleotides of NUMB exon
 9. 21. The antisense oligonucleotide of claim16, wherein the oligonucleotide is between 7 and 31 nucleotides long.22. The antisense oligonucleotide of claim 16, wherein theoligonucleotide does not comprise a sequence that is complementary tothe 5′ and/or 3′ exon-intron junction of exon
 9. 23. The antisenseoligonucleotide of claim 16, wherein the oligonucleotide is an RNA or amodified RNA molecule, a DNA or a modified DNA molecule, or a mixture ofnative or modified RNA and native or modified RNA.
 24. The antisenseoligonucleotide of claim 23, wherein the modified RNA or DNA moleculecomprises one or more chemical modifications selected from the groupconsisting of: a locked nucleic acid, a phosphorothioate linkage, a2′-O-methylated nucleotide, a 2′-O-methoxyethyl modified (2′MOE)nucleotide, methylated cytosine, a constrained ethyl (cET) nucleic acid,a bridged nucleic acid (BNA), a phosphorodiamidate morpholino oligomer(PMO), a peptide nucleic acid (PNA), a cyclohexene nucleic acid (CeNA),a tricycle-DNA (tcDNA), N3′-P5′ phosphoroamidate (NP),2′-fluoro-2′-deoxyadenosine-5′-triphosphate,2′-fluoro-2′-deoxycytidine-5′-triphosphate,2′-fluoro-2′-deoxyguanosine-5′-triphosphate,2′-fluoro-2′-deoxyuridine-5′-triphosphate,2′-fluoro-2′-deoxythymidine-5′-triphosphate,methyladenosine-5′-triphosphate, 2′-O-methylcytidine-5′-triphosphate,2′-O-methylguanosine-5′-triphosphate,2′-O-methyluridine-5′-triphosphate, 2′-O-methylinosine-5′-triphosphate,2′-O-methyl-2-2′-O-methyl-5-2′-O-2′-O-methylpseudouridine-5′-triphosphate,and methyluridine-5′-triphosphate.
 25. The antisense oligonucleotide ofclaim 16, which is between 7 and 31 nucleotides long, and wherein theoligonucleotide is a locked nucleic acid.
 26. The antisenseoligonucleotide of claim 16, which is between 7 and 31 nucleotides longand wherein the oligonucleotide is a 2′-O-methyl phosphorothioateribonucleic acid; or which is between 7 and 31 nucleotides long andwherein the oligonucleotide is a 2′-O-methoxyethyl-modifiedphosphorothioate ribonucleic acid.
 27. The antisense oligonucleotide ofclaim 26, which is between 18 and 25 nucleotides long, and wherein theoligonucleotide is a 2′-O-methyl phosphorothioate ribonucleic acid. 28.The antisense oligonucleotide of claim 26, wherein all of thenucleotides of the antisense oligonucleotide are 2′-O-methylatednucleotides.
 29. The antisense oligonucleotide of claim 26, which isbetween 18 and 25 nucleotides long, and wherein the oligonucleotide is a2′-O-methoxyethyl-modified phosphorothioate ribonucleic acid.
 30. Theantisense oligonucleotide of claim 26, wherein all of the nucleotides ofthe antisense oligonucleotide are 2′-O-methoxyethylated nucleotides. 31.The oligonucleotide of claim 16, wherein at least one cytosine residuewithin the oligonucleotide is methylated.
 32. The antisenseoligonucleotide of claim 16, wherein the oligonucleotide comprises asequence selected from: (a) (SEQ ID NO: 36) AGGCUGA, (SEQ ID NO: 37)GUCAGUG, (SEQ ID NO: 38) CCAUUAG, (SEQ ID NO: 39) CUGAGUC, or(SEQ ID NO: 40) AGUGCCA; and/or (b) (SEQ ID NO: 41) AGGCUGAGUCAGUG, or(SEQ ID NO: 42) GUCAGUGCCAUUAG; and/or (c) (SEQ ID NO: 43)AGGCUGAGUCAGUGCCAUUAG or (SEQ ID NO: 12) AGGCUGAGUCAGUGCCAUUAG; and/or(d) (SEQ ID NO: 22) AGGGUUGGUUUCACGCACAGG or (SEQ ID NO: 24)GGCUGCAAUUUCCUUGUUAGC; and/or (e) (SEQ ID NO: 19) CACAUGGAAGGCUGAGUCAGU;(SEQ ID NO: 13) GCUUAGCAAGCACAUGGAAGG; and (SEQ ID NO: 20)AGUAUGGGCUGGCUUAGCAAG


33. An antisense oligonucleotide for reducing inclusion of NUMB exon 9in a population of mature NUMB transcripts according to claim 16, theantisense oligonucleotide comprising a sequence of 19 nucleotides thatis complementary to a target region of a NUMB transcript; wherein thetarget region is comprised within: the sequence (SEQ ID NO: 10)CCGUAGCAAUGCCUGUGCGUGAAACCAA CCCUUGGGCCCAUG; or the sequence(SEQ. ID NO: 44) CUAAUGGCACUGACUCAGCCU; or the sequence (SEQ ID NO: 31)CCUGUGCGUGAAACCAACCCU; or the sequence (SEQ ID NO: 32)GCUAACAAGGAAAUUGCAGCC; or the sequence (SEQ ID NO: 5)CCGUAGCAAUGCCUGUGCGUG; or the sequence (SEQ ID NO: 6)AAACCAACCCUUGGGCCCAUG; or the sequence (SEQ ID NO: 35)UGGGCCCAUGCCCCUGAUGCU


34. An antisense oligonucleotide for reducing inclusion of NUMB exon 9in a population of mature NUMB transcripts according to claim 16, theantisense oligonucleotide comprising a sequence of 20 nucleotides thatis complementary to a target region of a NUMB transcript; wherein thetarget region is comprised within: the sequence (SEQ ID NO: 10)CCGUAGCAAUGCCUGUGCGUGAAACCAACCCUUGGGCCCAUG; or the sequence(SEQ. ID NO: 44) CUAAUGGCACUGACUCAGCCU; or the sequence (SEQ ID NO: 31)CCUGUGCGUGAAACCAACCCU; or the sequence (SEQ ID NO: 32)GCUAACAAGGAAAUUGCAGCC; or the sequence (SEQ ID NO: 5)CCGUAGCAAUGCCUGUGCGUG; or the sequence (SEQ ID NO: 6)AAACCAACCCUUGGGCCCAUG; or the sequence (SEQ ID NO: 35)UGGGCCCAUGCCCCUGAUGCU; or the sequence (SEQ ID NO: 305)CUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCU AAGCCAGCCCAUACU; or the sequence(SEQ ID NO: 9) CUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGC.


35. An antisense oligonucleotide for reducing inclusion of NUMB exon 9in a population of mature NUMB transcripts according to claim 16, theantisense oligonucleotide comprising a sequence of at least 21nucleotides that is complementary to a target region of a NUMBtranscript; wherein the target region is comprised within: the sequence(SEQ ID NO: 10) CCGUAGCAAUGCCUGUGCGUGAAACCAACCCUUGGGCCCAUG; orthe sequence (SEQ. ID NO: 44) CUAAUGGCACUGACUCAGCCU; or the sequence(SEQ ID NO: 31) CCUGUGCGUGAAACCAACCCU; or the sequence (SEQ ID NO: 32)GCUAACAAGGAAAUUGCAGCC; or the sequence (SEQ ID NO: 5)CCGUAGCAAUGCCUGUGCGUG; or the sequence (SEQ ID NO: 6)AAACCAACCCUUGGGCCCAUG; or the sequence (SEQ ID NO: 35)UGGGCCCAUGCCCCUGAUGCU; or the sequence (SEQ ID NO: 305)CUAAUGGCACUGACUCAGCCUUCCAUGU GCUUGCUAAGCCAGCCCAUACU; or the sequence(SEQ ID NO: 9) CUAAUGGCACUGACUCAGCCUUCCAUGUGCUUGCUAAGC.


36. A pharmaceutical composition comprising: one or more antisenseoligonucleotides according to claim 16; and one or morepharmaceutically-acceptable excipients.